Pyrazolopyrimidone derivatives and methods of use thereof

ABSTRACT

Pyrazolopyrimidone derivatives and methods of use thereof are described. Specifically, pharmaceutical uses of pyrazolopyrimidone derivatives represented by the general formula (II) and pharmaceutically acceptable salts thereof are described, wherein the definitions of substituents in the general formula (II) are the same as the definitions in the specification. The pyrazolopyrimidone derivatives are useful as gonadotropin releasing hormone (GnRH) antagonists, such as for therapeutic agents for endometriosis.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation of co-pending U.S. patent applicationSer. No. 15/969,098, filed on May 2, 2018, which is a Divisional of U.S.patent application Ser. No. 15/031,410, filed on Apr. 22, 2016 andissued on Jun. 26, 2018 as U.S. Pat. No. 10,005,781, which is a Section371 of International Application No. PCT/CN2014/087906, filed Sep. 30,2014, which was published in the Chinese language on May 7, 2015, underInternational Publication No. WO 2015/062391 A1, which claims priorityunder 35 U.S.C. § 119(b) to Chinese Application No. 201310525956.9,filed Oct. 30, 2013, the disclosures of which are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a novel pyrazolopyrimidone derivativeor pyrrolotriazone derivative and a pharmaceutically acceptable saltthereof, a preparation method thereof, and a pharmaceutical compositioncontaining the same, as well as use of the same as agonadotropin-releasing hormone (GnRH) receptor antagonist, particularlyas a therapeutic agent for the diseases such as endometriosis.

BACKGROUND OF THE INVENTION

Endometriosis is a common estrogen-dependent gynecological disease,which often occurs in women of childbearing-age, though the mechanism isunclear. The difficult diagnosis and unclear pathogenesis ofendometriosis severely hinder the discovery of effective treatments. Atpresent, endometriosis is mainly diagnosed by laparoscopy, and treatedby surgery, or controlled by taking contraceptives, GnRH receptoragonists, or progestogen to reduce body estrogen levels.

Presently, the incidence rate of endometriosis is high. Statistical datafrom Datamonitor 2009 shows that the number of female patients sufferingfrom endometriosis has been more than 68 million in only two countries(India and China) (31,288,000 in India, 37,535,000 in China), while thedata in the seven major markets has been more than 17 million.Datamonitor expects that during 2009-2018, the endometriosis drug marketwill grow from $764 million in 2009 ($596 million in US, $117 million inEuropean Union, $51 million in Japan) to $1.156 billion in 2018 ($844million in US, $206 million in European Union, $106 million in Japan),and the growing space in the Chinese market will be larger.

Gonadotropin-releasing hormone (Gonadoliberin; GnRH), also known asluteinizing hormone releasing hormone (LHRH), is a decapeptide hormone(pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) synthesized byhypothalamic neuroendocrine cells, and is a central regulatory factor ofthe endocrine reproductive system. GnRH plays an important role in thehypothalamus-pituitary-gonadal axis system by being transported to thepituitary through the hypothalamic pituitary portal circulation systemand then binding to GnRH receptor cells in the anterior pituitary, bypromoting the secretion and release of gonadotropin luteinizinghormones, such as Luteinizing Hormone (LH) and follicle-stimulatinghormone (FSH), and by regulating normal development of the ovary andcorpus luteum. GnRH receptor plays its regulatory role by coupling withG protein, which is capable of activating the calciumphosphatidylinositol second messenger system. LH regulates theproduction of sex steroids, and FSH regulates male spermatogenesis andmaturation of female ovarian follicles.

LH and FSH are released into circulation, and bind to receptors on thespecific cells of ovaries or testes to stimulate steroid production.Diseases such as endometriosis, uterine leiomyoma, and prostate cancerare aggravated in the presence of sex steroids. It is necessary toadminister long-acting peptide GnRH receptor agonists and antagonists tocontrol the diseases.

Peptide GnRH receptor antagonists include GnRH-derived linear peptides(U.S. Pat. No. 5,171,835), cyclic hexapeptide derivatives (US2002/0065309), bicyclic peptide derivatives (Journal of MedicinalChemistry, 1993; 36: 3265-73), etc.; and peptide GnRH receptor agonistsinclude Leuprorelin (pGlu-His-Trp-Ser-Tyr-d-Leu-Leu-Arg-Pro-NHEt).However, there are many unresolved issues for peptide compoundsincluding oral absorption, dosage form, dose volume, drug stability,sustained action, and metabolic stability and the like. The primaryreason that small molecule GnRH receptor antagonist therapy is superiorto the existing peptide-based therapy is largely due to the oraladministration of small molecule GnRH receptor antagonists, which isconvenient and efficient. Studies have shown that small moleculeantagonists have a significant efficacy for hormone-dependent diseasessuch as endometriosis, precocious puberty, and prostate cancer.

An indirect tumor inhibition mechanism mediated by GnRH agonists is thatGnRH agonists decrease the secretion of sex hormones and then indirectlyinhibit the growth of tumor cells by long-term effects on thehypothalamic-pituitary-gonadal axis, which leads to a reduction ofpituitary gonadotropins (FSH, LH), whereas GnRH receptor antagonistsdirectly inhibit the release of pituitary gonadotropins, and theninhibit the growth of tumor cells.

In view of the limitations of peptide GnRH receptor antagonists, certainnon-peptide GnRH receptor antagonists have been proposed and put intodevelopment, clinical trials, and marketing stages. For example,Elagolix (also known as NBI-56418 or ABT-620), is a small molecule GnRHreceptor antagonist co-developed by Abbott and Neurocrine BiosciencesInc, which is currently in phase III clinical stage, and mainly used forthe treatment of endometriosis (phase III) and uterine leiomyoma (phaseII). In June 2012, the data from the endometriosis phase II trial werepresented at the 94th Annual Meeting of the Endocrine Society inHouston. Among the 131 women suffering from endometriosis who weretreated with elagolix (150 or 250 mg qd), leuprorelin depot (3.75 mg sc,once a month for 12 weeks) or placebo, the serum estrogen level in thepatients of the elagolix treatment group was lower than that of theleuprorelin treatment group and placebo group. Meanwhile, the safety andtolerability of elagolix has been well verified.

Relugolix, also known as TAK-385, is an oral small molecule GnRHreceptor antagonist developed by Takeda Pharmaceutical in Japan, whichis used for the treatment of endometriosis, uterine leiomyoma, andprostate cancer. The study of endometriosis and uterine leiomyoma hasbeen in phase II clinical trials since 2011, and the study of prostatecancer has been in phase I clinical trials since the same year.

At present, a series of patent applications concerning small moleculeGnRH receptor antagonists have been published, including WO2006096785,WO2010026993, WO2011076687, WO2012175514, etc.

Despite numerous significant studies that have been conducted in thisfield, there remains a need to develop more effective small moleculeGnRH receptor antagonists.

The present disclosure provides structurally new GnRH receptorantagonists, and it is found that compounds having such structures havegood activity, and exhibit excellent treatment effects for endocrinereproductive system diseases.

SUMMARY OF THE INVENTION

The present invention is directed to provide a compound of formula (I),or a tautomer, mesomer, racemate, enantiomer, or diastereomer thereof,or a mixture thereof, or a pharmaceutically acceptable salt thereof:

wherein:when G is N, D is C and E is —CH—;when G is C, D and E are N;R¹ is selected from the group consisting of alkyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, and —OR⁶, wherein the alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl are each independently and optionallyfurther substituted with one or more groups selected from the groupconsisting of halogen, cyano, nitro, alkyl, haloalkyl, hydroxyalkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, —OR⁶,—C(O)OR⁶, —OC(O)R⁶, —NHS(O)_(m)R⁶, —C(O)R⁶, —NHC(O)R⁶, —NR⁷R⁸,—OC(O)NR⁷R⁸, —C(O)NR⁷R⁸, —NHC(O)NHR⁶, —NHC(O)OR⁶, and —NHC(O)NHOR⁶;R² is alkyl, wherein the alkyl is further substituted with one or moregroups selected from aryl and heteroaryl, wherein the aryl andheteroaryl are each optionally further substituted with one or moregroups selected from the group consisting of halogen, alkyl, haloalkyl,cyano, nitro, —C(O)OR⁶, —C(O)NR⁷R⁸, —OC(O)NR⁷R⁸, —OR⁶, —NHS(O)_(m)R⁶,—NHC(O)R⁶, and —NR⁷R⁸, wherein the haloalkyl is preferablytrifluoromethyl;R³ is selected from the group consisting of aryl and heteroaryl, whereinthe aryl and heteroaryl are each optionally further substituted with oneor more groups selected from the group consisting of halogen, alkyl,haloalkyl, —OR⁶, —C(O)OR⁶, —OC(O)R⁶, —C(O)R⁶, —NR⁷R⁸, —OC(O)NR⁷R⁸,—C(O)NR⁷R⁸, —NHS(O)_(m)R⁶, —NHC(O)R⁶, —NHC(O)OR⁶, —NHC(O)NHR⁶, and—NHC(O)NHOR⁶;R⁴ is alkyl;R⁵ is selected from the group consisting of hydrogen, cycloalkyl,heterocyclyl, aryl, heteroaryl, —OR⁵, —NR⁷R⁸, and —NR⁷S(O)_(m)R⁶,wherein the alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl areeach optionally further substituted with one or more groups selectedfrom the group consisting of halogen, oxo, alkyl, haloalkyl,hydroxyalkyl, —OR⁶, —C(O)OR⁶, —OC(O)R⁶, —NR⁷S(O)_(m)R⁶, —S(O)_(m)R⁶,—C(O)R⁶, and —NHC(O)R⁶;R⁶ is selected from the group consisting of hydrogen, alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl, wherein the alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl are each independently and optionallyfurther substituted with one or more groups selected from the groupconsisting of alkyl, halogen, hydroxyl, alkoxy, cycloalkyl,heterocyclyl, aryl, heteroaryl, carboxylic acid, and carboxylic ester;R⁷ and R⁸ are each independently selected from the group consisting ofhydrogen, alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, whereinthe alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are eachindependently and optionally further substituted with one or more groupsselected from the group consisting of alkyl, halogen, hydroxy, alkoxy,cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxylic acid, andcarboxylic ester;or, R⁷ and R⁸ are taken together with the attached N atom to form aheterocyclyl, wherein the heterocyclyl contains one or more heteroatomsof N, O, or S(O)_(m), and the heterocyclyl is optionally furthersubstituted with one or more groups selected from the group consistingof alkyl, halogen, hydroxyl, alkoxy, cycloalkyl, heterocyclyl, aryl,heteroaryl, carboxylic acid, and carboxylic ester;m is 0, 1 or 2;n is 1, 2, 3 or 4; andp is 0, 1 or 2.

In a preferred embodiment of the present invention, the compound offormula (I), or a tautomer, mesomer, racemate, enantiomer, ordiastereomer thereof, or mixture thereof, or a pharmaceuticallyacceptable salt thereof, is a compound of formula (II), or a tautomer,mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or apharmaceutically acceptable salt thereof:

wherein: n, and R¹ to R⁵ are as defined in formula (I).

In another preferred embodiment of the present invention, the compoundof formula (I), or a tautomer, mesomer, racemate, enantiomer, ordiastereomer thereof, or mixture thereof, or a pharmaceuticallyacceptable salt thereof, is a compound of formula (III), or a tautomer,mesomer, racemate, enantiomer, or diastereomer thereof, or mixturethereof, or a pharmaceutically acceptable salt thereof:

wherein: n, and R¹ to R⁵ are as defined in formula (I).

In another preferred embodiment of the present invention, the compoundof formula (I), or a tautomer, mesomer, racemate, enantiomer, ordiastereomer thereof, or mixture thereof, or a pharmaceuticallyacceptable salt thereof, wherein R¹ is selected from the groupconsisting of aryl and heteroaryl, preferably phenyl and pyridazinyl,wherein the aryl and heteroaryl are each optionally further substitutedwith one or more groups selected from the group consisting of halogen,cyano, nitro, alkyl, haloalkyl, hydroxyalkyl, and —OR⁶.

In another preferred embodiment of the present invention, the compoundof formula (I), or a tautomer, mesomer, racemate, enantiomer, ordiastereomer thereof, or mixture thereof, or a pharmaceuticallyacceptable salt thereof, wherein R² is benzyl, wherein the benzyl isoptionally further substituted with one or more groups selected from thegroup consisting of halogen, alkyl, haloalkyl, cyano, nitro, and —OR⁶.

In another preferred embodiment of the present invention, the compoundof formula (I), or a tautomer, mesomer, racemate, enantiomer, ordiastereomer thereof, or mixture thereof, or a pharmaceuticallyacceptable salt thereof, wherein R³ is selected from aryl, preferablyphenyl, wherein the aryl is optionally further substituted with one ormore groups selected from the group consisting of —NHC(O)R⁶, —NHC(O)OR⁶,—NHC(O)NHR⁶ and —NHC(O)NHOR⁶, preferably —NHC(O)NHR⁶, and —NHC(O)NHOR⁶.

In another preferred embodiment of the present invention, the compoundof formula (I), or a tautomer, mesomer, racemate, enantiomer, ordiastereomer thereof, or mixture thereof, or a pharmaceuticallyacceptable salt thereof, wherein R⁴ is methyl.

In another preferred embodiment of the present invention, the compoundof formula (I), or a tautomer, mesomer, racemate, enantiomer, ordiastereomer thereof, or mixture thereof, or a pharmaceuticallyacceptable salt thereof, wherein R⁵ is hydrogen, and n is 1 or 2.

In another preferred embodiment of the present invention, the compoundof formula (I), or a tautomer, mesomer, racemate, enantiomer, ordiastereomer thereof, or mixture thereof, or a pharmaceuticallyacceptable salt thereof, is a compound of formula (IV), or a tautomer,mesomer, racemate, enantiomer, or diastereomer thereof, or mixturethereof, or a pharmaceutically acceptable salt thereof:

wherein: n, D, E, G, R¹, R², R⁴, and R⁵ are as defined in formula (I);R^(a) is selected from the group consisting of alkyl and —OR⁶, whereinthe alkyl is optionally further substituted with one or more groupsselected from the group consisting of alkyl, halogen, hydroxy, alkoxy,cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxylic acid, andcarboxylic ester; andR⁶ is alkyl, wherein the alkyl is optionally further substituted withone or more groups selected from the group consisting of alkyl, halogen,hydroxy, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxylicacid, and carboxylic ester.

Typical compounds of the present invention include, but are not limitedto, the following:

Example No. Structure and Name 1

N-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)acetamide 2

1-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxyurea 3

1-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-ethylurea 4

methyl(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)carbamate 5

1-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methylurea 6

1-(4-(3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-ethylurea 7

1-(4-(3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxyurea 8

1-(4-(3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methylurea 9

1-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methylurea 10

1-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-ethylurea 11

1-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxyurea 12

1-(4-(3-((dimethylamino)methyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-5-(6-methoxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methylurea 13

1-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-ethylurea 14

1-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-methylurea 15

1-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-methoxyurea 16

1-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-methylurea 17

1-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-hydroxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxyurea 18

1-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-methoxyureaor a tautomer, mesomer, racemate, enantiomer, or diastereomer thereof,or mixture thereof, or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a process forpreparing the compound of formula (I), or a tautomer, mesomer, racemate,enantiomer, or diastereomer thereof, or mixture thereof, or apharmaceutically acceptable salt thereof, comprising a step of:

reacting a compound of formula (IA) with the amine of NH(R⁴)(CH₂)nR⁵;optionally reducing and/or acylating the resulting product to obtain thecompound of formula (I); wherein: X is halogen; and n, D, E, G and R¹ toR⁵ are as defined in formula (I).

Reducing agents include, but are not limited to, hydrogen and ironpowder; Acylating agents include, but are not limited to, carboxylicacids, acyl chloride, sulfuryl chloride, halogenated methyl formate,isocyanate, triphosgene, and methoxyamine.

In another aspect, the present invention provides a process forpreparing the compound of formula (I), or a tautomer, mesomer, racemate,enantiomer, or diastereomer thereof, or mixture thereof, or apharmaceutically acceptable salt thereof, comprising a step of:

reacting a compound of formula (IB) with R²X in the presence of analkaline reagent; optionally reducing and/or acylating the resultingproduct to obtain the compound of formula (I);wherein: X is halogen, and n, D, E, G and R¹ to R⁵ are as defined informula (I).

Alkaline reagents include, but are not limited to, organic bases andinorganic bases, wherein the organic base includes, but is not limitedto, triethylamine, pyridine, 2,6-lutidine, sodium methoxide, lithiumhexamethyldisilazide, sodium hexamethyldisilazide, n-butyllithium,potassium tert-butoxide, and tetrabutyl ammonium bromide; the inorganicbase includes, but is not limited to, sodium hydride, sodium carbonate,sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesiumcarbonate, lithium hydroxide, sodium hydroxide, and potassium hydroxide,preferably potassium carbonate and sodium methoxide.

Reducing agents include, but are not limited to, hydrogen and ironpowder;

Acylating agents include, but are not limited to, carboxylic acids, acylchloride, sulfuryl chloride, halogenated methyl formate, isocyanate,triphosgene, and methoxyamine.

A compound of formula (IA) or formula (IB), or a pharmaceuticallyacceptable salt thereof, which is used as an intermediate in thepreparation of a compound of formula (I):

wherein: X is halogen, and n, D, E, G and R¹ to R⁵ are as defined informula (I).

A process for preparing the compound of formula (IA), or apharmaceutically acceptable salt thereof, comprising a step of:

performing a halogenation reaction of a compound of formula (Ib) toobtain the compound of formula (IA);wherein: D, E, G and R¹ to R³ are as defined in formula (I).

A process for preparing the compound of formula (IB), or apharmaceutically acceptable salt thereof, comprising a step of:

performing a cyclization reaction of a compound of formula (Ig) toobtain the compound of formula (IB);wherein: n, D, E, G, and R¹, R³ to R⁵ are as defined in formula (I); andR^(b) is alkyl.

Typical compounds of formula (IA) include, but are not limited to, thefollowing:

Exam- ple No. Structure and Name 1f

3-(bromomethyl)-7-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione 6b

3-(bromomethyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione 9e

3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione 12d

3-(bromomethyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-5-(6-methoxypyridazin-3-yl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione 13h

5-(bromomethyl)-1-(2,6-difluorobenzyl)-3-(2-fluoro-3-methoxyphenyl)-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione 16h

5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dioneor a tautomer, mesomer, racemate, enantiomer, or diastereomer thereof,or mixture thereof, or a pharmaceutically acceptable salt thereof.

A compound of formula (IVA), or a pharmaceutically acceptable saltthereof, which is used as an intermediate in the preparation of acompound of formula (IV):

wherein: n, D, E, G, R¹, R², R⁴, and R⁵ are as defined in formula (I).

Typical compounds of formula (IVA) include, but are not limited to thefollowing:

Exam- ple No. Structure and Name 1h

2-(4-aminophenyl)-7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione 6d

2-(4-aminophenyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione 9g

2-(4-aminophenyl)-7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione 12f

2-(4-aminophenyl)-3-((dimethylamino)methyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-5-(6-methoxypyridazin-3-yl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione 13j

6-(4-aminophenyl)-1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione 16j

6-(4-aminophenyl)-1-(2,6-ditluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dioneor a tautomer, mesomer, racemate, enantiomer, or diastereomer thereof,or mixture thereof, or a pharmaceutically acceptable salt thereof.

A process for preparing the compound of formula (IV), comprising a stepof:

further reacting the compound of formula (IVA) with an acylating agentto obtain a compound of formula (IV);wherein: n, D, E, G, R¹, R², R⁴, and R⁵ are as defined in formula (I).

Acylating agents include, but are not limited to, carboxylic acids, acylchloride, sulfuryl chloride, halogenated methyl formate, isocyanate,triphosgene, and methoxyamine.

The present invention further relates to a pharmaceutical composition,comprising a therapeutically effective amount of a compound of formula(I), or a tautomer, mesomer, racemate, enantiomer, or diastereomerthereof, or mixture thereof, or a pharmaceutically acceptable saltthereof, and one or more pharmaceutically acceptable carriers, diluents,or excipients.

The present invention further relates to use of a compound of formula(I), or a tautomer, mesomer, racemate, enantiomer, or diastereomerthereof, or mixture thereof, or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition comprising the same, in thepreparation of a medicament as a GnRH receptor antagonist.

The present invention further relates to use of a compound of formula(I), or a tautomer, mesomer, racemate, enantiomer, or diastereomerthereof, or mixture thereof, or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition comprising the same, in thepreparation of a medicament for the treatment of gonadal hormone-relateddiseases.

The present invention further relates to use of a compound of formula(I), or a tautomer, mesomer, racemate, enantiomer, or diastereomerthereof, or mixture thereof, or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition comprising the same, in thepreparation of medicaments for the treatment or prevention of sexhormone-dependent cancers (e.g., prostatic cancer, uterine cancer,breast cancer, pituitary cancer, etc.), bone metastasis of sexhormone-dependent cancers, prostatic hyperplasia, uterine leiomyoma,endometriosis, uterine fibroids, precocious puberty, amenorrhea,premenstrual syndrome, dysmenorrhea, multilocular ovary syndrome,polycystic ovary syndrome, acne, alopecia, Alzheimer's disease,infertility, irritable bowel syndrome, benign or malignant tumors whichare hormone-independent and LH-RH sensitive, or flush; or in thepreparation of a reproductive regulator, contraceptive, or an ovulationstimulant; or in the preparation of medicaments for the treatment orprevention of postoperative recurrence of sex hormone-dependent cancer,preferably prostate cancer, uterine cancer, breast cancer, endometriosisor uterine leiomyoma.

The present invention further relates to a method for inhibiting GnRHreceptor activity, comprising administering to a subject in need thereofa therapeutically effective amount of a compound of formula (I), or atautomer, mesomer, racemate, enantiomer, or diastereomer thereof, ormixture thereof, or a pharmaceutically acceptable salt thereof, or apharmaceutical composition containing the same.

The present invention further relates to a method for treating a gonadalhormone-related disease, comprising administering to a subject in needthereof a therapeutically effective amount of a compound of formula (I),or a tautomer, mesomer, racemate, enantiomer, or diastereomer thereof,or mixture thereof, or a pharmaceutically acceptable salt thereof, or apharmaceutical composition containing the same.

The present invention further relates to a method for the prevention ortreatment of sex hormone-dependent cancers (prostate cancer, uterinecancer, breast cancer, pituitary cancer, etc.), bone metastatic sexhormone-dependent cancer, prostatic hyperplasia, uterine leiomyoma,endometriosis, uterine fibroids, precocious puberty, amenorrhea,premenstrual syndrome, dysmenorrhea, multilocular ovary syndrome,polycystic ovary syndrome, acne, alopecia, Alzheimer's disease,infertility, irritable bowel syndrome, benign or malignant tumors whichare hormone-independent and LH-RH sensitive, or flush, preferablyprostate cancer, uterine cancer, breast cancer, endometriosis, oruterine leiomyoma; or for regulating reproduction, contraception orstimulating ovulation; or for the prevention of postoperative recurrenceof sex hormone-dependent cancer.

The present invention also relates to a compound of formula (I), or atautomer, mesomer, racemate, enantiomer, or diastereomer thereof, ormixture thereof, or a pharmaceutically acceptable salt thereof, or apharmaceutical composition containing the same, for use as a medicamentfor inhibiting the activity of GnRH receptor.

The present invention also relates to a compound of the formula (I), ora tautomer, mesomer, racemate, enantiomer, or diastereomer thereof, ormixture thereof, or a pharmaceutically acceptable salt thereof, or apharmaceutical composition containing the same, for use as a medicamentfor the treatment of sex hormone-related diseases.

The present invention further relates to a compound of formula (I), or atautomer, mesomer, racemate, enantiomer, or diastereomer thereof, ormixture thereof, or a pharmaceutically acceptable salt thereof, or apharmaceutical composition containing the same, for use as apreventative or regulatory agent for hormone-dependent diseases,particularly; or for use as a preventative or therapeutic agent for sexhormone-dependent cancers (e.g., prostatic cancer, uterine cancer,breast cancer, pituitary cancer, etc.), prostatic hyperplasia, uterineleiomyoma, endometriosis, uterine fibroids, precocious puberty,amenorrhea syndrome, premenstrual syndrome, multilocular ovary syndrome,acne, alopecia, Alzheimer's disease, etc.; or for use as a pregnancyregulator (contraceptive and the like), infertility therapeutic agent,or menstruation regulator; or for use as a preventative or therapeuticagent for irritable bowel syndrome; or for use as a preventative agentfor post-operative recurrence of sex hormone-dependent cancer, and thelike; preferably prostate cancer, uterine cancer, breast cancer,endometriosis, or uterine leiomyoma.

The pharmaceutical compositions containing the active ingredient can bein a form suitable for oral administration, such as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Oralcompositions can be prepared according to any known method for thepreparation of pharmaceutical compositions in the art. Such compositionscan contain one or more additives selected from the group consisting ofsweetener, flavoring agents, colorant, and preservatives, to provide apleasing and palatable pharmaceutical formulation. Tablets contain theactive ingredient and nontoxic pharmaceutically acceptable excipientssuitable for the manufacture of tablets. These excipients can be inertexcipients, such as calcium carbonate, sodium carbonate, lactose,calcium phosphate or sodium phosphate; granulating and disintegratingagents, such as microcrystalline cellulose, cross-linked sodiumcarboxymethyl cellulose, corn starch or alginic acid; binder such asstarch, gelatin, polyvinylpyrrolidone or acacia; and lubricant, such asmagnesium stearate, stearic acid or talc. The tablets can be uncoated orcoated by means of known techniques which can mask drug taste or delaythe disintegration and absorption of a drug in the gastrointestinaltract, thereby providing sustained release over an extended period. Forexample, the water soluble taste masking material which can be used inthe present invention is such as hydroxypropyl methylcellulose orhydroxypropyl cellulose, or the sustained release material which can beused in the present invention is such as ethyl cellulose, celluloseacetate butyrate.

Oral formulations can also be provided as hard gelatin capsules in whichthe active ingredient is mixed with an inert solid diluent, such ascalcium carbonate, calcium phosphate or kaolin, or as soft gelatincapsules in which the active ingredient is mixed with a water solublecarrier, such as polyethyleneglycol or an oil medium, for example peanutoil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active ingredient in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose, sodium alginate,polyvinylpyrrolidone, and gum acacia; dispersants or humectants, whichmay be a naturally occurring phosphatide, such as lecithin, orcondensation products of an alkylene oxide with fatty acids, such aspolyoxyethylene stearate, or condensation products of ethylene oxidewith long chain aliphatic alcohol, such as heptadecaethyleneoxy cetanol,or condensation products of ethylene oxide with part esters derived fromfatty acids and hexitols, such as polyoxyethylene sorbitol monooleate,or condensation products of ethylene oxide with partial esters derivedfrom fatty acids and hexitol anhydrides, such as polyoxyethylenesorbitan monooleate. The aqueous suspension can also contain one or morepreservatives, such as ethylparaben or n-propylparaben, one or morecolorants, one or more flavoring agents, and one or more sweeteners,such as sucrose, saccharin or aspartame.

Oil suspensions can be formulated by suspending the active ingredient ina vegetable oil, such as Arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil, such as liquid paraffin. The oil suspensioncan contain a thickener, such as beeswax, hard paraffin or cetylalcohol. The aforesaid sweetener and flavoring agents can be added toprovide a palatable preparation. These compositions can be preserved bythe addition of an antioxidant, such as butylated hydroxyanisole orα-tocopherol.

Dispersible powders and granules suitable for the preparation of anaqueous suspension by the addition of water provide the activeingredient in admixture with a dispersant or wetting agent, suspendingagent, or one or more preservatives. Suitable dispersants or wettingagents and suspending agents are exemplified by those already mentionedabove. Additional excipients, such as sweeteners, flavoring agents, andcolorants, can also be added. These compositions can be preserved by theaddition of an antioxidant, such as ascorbic acid.

The present pharmaceutical composition can also be in the form of anoil-in-water emulsion. The oil phase can be a vegetable oil, such asolive oil or Arachis oil, or a mineral oil, such as liquid paraffin, ora mixture thereof. Suitable emulsifying agents can be naturallyoccurring phosphatides, such as soy bean lecithin, and esters or partialesters derived from fatty acids and hexitol anhydrides, such as sorbitanmonooleate, and condensation products of the said partial esters withethylene oxide, such as polyoxyethylene sorbitol monooleate. Theemulsion can also contain sweeteners, flavoring agents, preservatives,and antioxidants. Syrups and elixirs can be formulated with sweetener,such as glycerol, propylene glycol, sorbitol or sucrose. Suchformulations can also contain a demulcent, a preservative, a colorant,or an antioxidant.

The pharmaceutical composition can be in the form of a sterileinjectable aqueous solution. The acceptable vehicles and solvents thatcan be employed are water, Ringer's solution and isotonic sodiumchloride solution. The sterile injectable preparation can also be asterile injectable oil-in-water microemulsion in which the activeingredient is dissolved in the oil phase. For example, the activeingredient can be first dissolved in a mixture of soybean oil andlecithin, the oil solution then is introduced into a mixture of waterand glycerol and processed to form a microemulsion. The injectablesolutions or microemulsions can be introduced into an individual'sbloodstream by local bolus injection. Alternatively, it may beadvantageous to administer the solution or microemulsion in such a wayas to maintain a constant circulating concentration of the presentcompound. In order to maintain such a constant concentration, acontinuous intravenous delivery device may be utilized. An example ofsuch device is Deltec CADD-PLUS™ 5400 intravenous injection pump.

The pharmaceutical composition can be in the form of a sterileinjectable aqueous or oily suspension for intramuscular or subcutaneousadministration. Such suspension can be formulated with suitabledispersant or wetting agents and suspending agents as described aboveaccording to known techniques. The sterile injectable preparation canalso be a sterile injectable solution or suspension prepared in anontoxic parenterally acceptable diluent or solvent, for example, asolution prepared in 1,3-butanediol. Moreover, a sterile fixed oil caneasily be used as a solvent or suspending medium. For this purpose, anysuitable fixed oils including synthetic mono- or di-glyceride can beemployed. Moreover, fatty acids such as oleic acid can be employed inthe preparation of injectables.

The present compound can be administered in the form of suppositoriesfor rectal administration. These pharmaceutical compositions can beprepared by mixing drug with a suitable non-irritating excipient whichis solid at regular room temperatures but liquid in the rectum, therebymelting in the rectum to release the drug. Such materials include cocoabutter, glycerinated gelatin, hydrogenated vegetable oils, mixture ofpolyethylene glycols and fatty acid esters of polyethylene glycol withvarious molecular weight.

It is well known to those skilled in the art that the dosage of a drugdepends on a variety of factors including, but are not limited to, thefollowing factors: activity of a particular compound, age of thepatient, weight of the patient, general health of the patient, behaviorof the patient, diet of the patient, time of administration, route ofadministration, rate of excretion, drug combination and the like. Inaddition, the best treatment, such as treatment model, daily dose of thecompound of formula (I) or the type of pharmaceutically acceptable saltthereof can be verified by the traditional therapeutic regimen.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise stated, the terms used in the specification and claimshave the meanings described below.

“Alkyl” refers to a saturated aliphatic hydrocarbon group includingC₁-C₂₀ linear and branched chain groups, preferably an alkyl having 1 to10 carbon atoms, more preferably an alkyl having 1 to 6 carbon atoms,even more preferably an alkyl having 1 to 4 carbon atoms, and mostpreferably methyl. Representative examples include, but are not limitedto, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl,sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl,2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl,n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl,1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl,1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl,4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl,3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl,2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl,2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl,2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl,3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl,4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl,2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl,n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and branched isomersthereof. More preferably, an alkyl group is a lower alkyl having 1 to 6carbon atoms. Representative examples include, but are not limited to,methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl,sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl,2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl,n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl,1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl,1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl,4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group can besubstituted or unsubstituted. When substituted, the substituent group(s)can be substituted at any available connection point, and thesubstituent group(s) is preferably one or more groups independentlyselected from the group consisting of alkyl, alkenyl, alkynyl,alkyloxyl, alkylthiol, alkylamino, halogen, thiol, hydroxy, nitro,cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy,heterocyclic alkoxy, cycloalkylthio, heterocycloalkylthio, oxo, amino,haloalkyl, hydroxyalkyl, carboxyl, alkoxycarbonyl, —OR⁶, —C(O)OR⁶,—OC(O)R⁶, —NHS(O)_(m)R⁶, —C(O)R⁶, —NHC(O)R⁶, —NHC(O)OR⁶, —NR⁷R⁸,—OC(O)NR⁷R⁸, —C(O)NR⁷R⁸, —NHC(O)NHR⁶ and —NHC(O)NHOR⁶.

“Alkenyl” refers to an alkyl defined as above that has at least twocarbon atoms and at least one carbon-carbon double bond, for example,vinyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, etc., preferablyC₂₋₁₀ alkenyl, more preferably C₂₋₆ alkenyl, and most preferably C₂₋₄alkenyl. The alkenyl group can be substituted or unsubstituted. Whensubstituted, the substituent group(s) is preferably one or more group(s)independently selected from the group consisting of alkyl, alkenyl,alkynyl, alkoxy, alkylthiol, alkylamino, halogen, thiol, hydroxy, nitro,cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy,heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, amino,haloalkyl, hydroxyalkyl, carboxyl, alkoxycarbonyl, —OR⁶, —C(O)OR⁶,—OC(O)R⁶, —NHS(O)_(m)R⁶, —C(O)R⁶, —NHC(O)R⁶, —NHC(O)OR⁶, —NR⁷R⁸,—OC(O)NR⁷R⁸, —C(O)NR⁷R⁸, —NHC(O)NHR⁶ and —NHC(O)NHOR⁶.

“Alkynyl” refers to an alkyl defined as above that has at least twocarbon atoms and at least one carbon-carbon triple bond, for example,ethynyl, 1-propynyl, 2-propynyl, 1-, 2-, or 3-butynyl, etc., preferablyC₂₋₁₀ alkynyl, more preferably C₂₋₆ alkynyl, and most preferably C₂₋₄alkynyl. The alkynyl group can be substituted or unsubstituted. Whensubstituted, the substituent group(s) is preferably one or more group(s)independently selected from the group consisting of alkyl, alkenyl,alkynyl, alkoxy, alkylthiol, alkylamino, halogen, thiol, hydroxy, nitro,cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy,heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, amino,haloalkyl, hydroxyalkyl, carboxyl, alkoxycarbonyl, —OR⁶, —C(O)OR⁶,—OC(O)R⁶, —NHS(O)_(m)R⁶, —C(O)R⁶, —NHC(O)R⁶, —NHC(O)OR⁶, —NR⁷R⁸,—OC(O)NR⁷R⁸, —C(O)NR⁷R⁸, —NHC(O)NHR⁶ and —NHC(O)NHOR⁶.

“Cycloalkyl” refers to a saturated or partially unsaturated monocyclicor polycyclic hydrocarbon group having 3 to 20 carbon atoms, preferably3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, even morepreferably 3 to 6 carbon atoms, and most preferably cyclopropyl.Representative examples of monocyclic cycloalkyl include, but are notlimited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl,cycloheptatrienyl, cyclooctyl, etc., preferably cyclopropyl, orcyclohexenyl. Polycyclic cycloalkyl includes a cycloalkyl having a spiroring, fused ring or bridged ring. The cycloalkyl group can besubstituted or unsubstituted. When substituted, the substituent group(s)is preferably one or more groups independently selected from the groupconsisting of alkyl, alkenyl, alkynyl, alkyloxyl, alkylthiol,alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl,heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy,cycloalkylthio, heterocycloalkylthio, oxo, amino, haloalkyl,hydroxyalkyl, carboxyl, alkoxycarbonyl, —OR⁶, —C(O)OR⁶, —OC(O)R⁶,—NHS(O)_(m)R⁶, —C(O)R⁶, —NHC(O)R⁶, —NHC(O)OR⁶, —NR⁷R⁸, —OC(O)NR⁷R⁸,—C(O)NR⁷R⁸, —NHC(O)NHR⁶ and —NHC(O)NHOR⁶.

“Heterocyclyl” refers to a 3 to 20 membered saturated or partiallyunsaturated monocyclic or polycyclic hydrocarbon group having one ormore heteroatoms selected from the group consisting of N, O, andS(O)_(m) (wherein m is an integer between 0 and 2) as ring atoms, butexcluding —O—O—, —O—S— or —S—S— in the ring, with the remaining ringatoms being C. A heterocyclyl preferably has 3 to 12 ring atoms, wherein1 to 4 atoms are heteroatoms; more preferably 3 to 10 ring atoms; andmost preferably 5 to 6 ring atoms. Representative examples of monocyclicheterocyclyl include, but are not limited to, pyrrolidinyl, piperidyl,piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, pyranyl,tetrahydrofuranyl, and the like. Polycyclic heterocyclyl includes theheterocyclyl having a spiro ring, fused ring or bridged ring. Theheterocyclyl group can be substituted or unsubstituted. Whensubstituted, the substituent group(s) is preferably one or more groupsindependently selected from the group consisting of alkyl, alkenyl,alkynyl, alkyloxyl, alkylthiol, alkylamino, halogen, thiol, hydroxy,nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy,heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, amino,haloalkyl, hydroxyalkyl, carboxyl, alkoxycarbonyl, —OR⁶, —C(O)OR⁶,—OC(O)R⁶, —NHS(O)_(m)R⁶, —C(O)R⁶, —NHC(O)R⁶, —NHC(O)OR⁶, —NR⁷R⁸,—OC(O)NR⁷R⁸, —C(O)NR⁷R⁸, —NHC(O)NHR⁶ and —NHC(O)NHOR⁶.

“Aryl” refers to a 6 to 14 membered all-carbon monocyclic ring orpolycyclic fused ring (a “fused” ring system means that each ring sharesan adjacent pair of carbon atoms with another ring in the system), whichhas a completely conjugated π-electron system. An aryl is preferably 6to 10 membered, more preferably phenyl and naphthyl, and most preferablyphenyl. The aryl can be fused to the ring of a heteroaryl, heterocyclylor cycloalkyl, wherein the ring bound to the parent structure is aryl.Representative examples include, but are not limited to, the followinggroups:

The aryl group can be substituted or unsubstituted. When substituted,the substituent group(s) is preferably one or more groups independentlyselected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy,alkylthiol, alkylamino, halogen, thiol, hydroxy, nitro, cyano,cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy,heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, —OR⁶, —C(O)OR⁶,—OC(O)R⁶, —NHS(O)_(m)R⁶, —C(O)R⁶, —NHC(O)R⁶, —NHC(O)OR⁶, —NR⁷R⁸,—OC(O)NR⁷R⁸, —C(O)NR⁷R⁸, —NHC(O)NHR⁶ and —NHC(O)NHOR⁶.

“Heteroaryl” refers to 5 to 14 membered all-carbon monocyclic ring orpolycyclic fused ring having 1 to 4 heteroatoms selected from the groupconsisting of O, S, and N, which has a completely conjugated π-electronsystem. A heteroaryl is preferably 5- to 10-membered, more preferably 5-or 6-membered, and most preferably furyl, thienyl, pyridinyl, pyrrolyl,N-alkyl pyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, andthe like. The heteroaryl can be fused with the ring of an aryl,heterocyclyl or cycloalkyl, wherein the ring bound to the parentstructure is heteroaryl. Representative examples include, but are notlimited to, the following groups:

The heteroaryl group can be substituted or unsubstituted. Whensubstituted, the substituent group(s) is preferably one or more groupsindependently selected from the group consisting of alkyl, alkenyl,alkynyl, alkoxy, alkylthiol, alkylamino, halogen, thiol, hydroxy, nitro,cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy,heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, —OR⁶, —C(O)OR⁶,—OC(O)R⁶, —NHS(O)_(m)R⁶, —C(O)R⁶, —NHC(O)R⁶, —NHC(O)OR⁶, —NR⁷R⁸,—OC(O)NR⁷R⁸, —C(O)NR⁷R⁸, —NHC(O)NHR⁶ and —NHC(O)NHOR⁶.

“Alkoxy” refers to both an —O-(alkyl) and an —O-(unsubstitutedcycloalkyl) group, wherein the alkyl and cycloalkyl are defined asabove. Representative examples include, but are not limited to, methoxy,ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy,cyclohexyloxy, and the like. The alkoxy can be substituted orunsubstituted. When substituted, the substituent is preferably one ormore group(s) independently selected from the group consisting of alkyl,alkenyl, alkynyl, alkoxy, alkylthiol, alkylamino, halogen, thiol,hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl,cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio,amino, haloalkyl, hydroxyalkyl, carboxyl, alkoxycarbonyl, —OR⁶,—C(O)OR⁶, —OC(O)R⁶, —NHS(O)_(m)R⁶, —C(O)R⁶, —NHC(O)R⁶, —NHC(O)OR⁶,—NR⁷R⁸, —OC(O)NR⁷R⁸, —C(O)NR⁷R⁸, —NHC(O)NHR⁶ and —NHC(O)NHOR⁶.

“Haloalkyl” refers to an alkyl group substituted by one or morehalogens, wherein the alkyl is as defined above.

“Hydroxy” refers to an —OH group.

“Hydroxy alkyl” refers to an alkyl group substituted by a hydroxy group,wherein the alkyl is as defined above.

“Halogen” refers to fluorine, chlorine, bromine, or bromine.

“Amino” refers to an —NH₂ group.

“Cyano” refers to a —CN group.

“Nitro” refers to a —NO₂ group.

“Oxo group” refers to a ═O group.

“Carboxylic acid” refers to an (alkyl) or (cycloalkyl) —C(O)OH.

“Sulfuryl chloride” refers to an (alkyl) or (cycloalkyl) —S(O)m-X(halo).

“Isocyanate” refers to an (alkyl) or (cycloalkyl) —N═C═O.

“Triphosgene” refers to a bis(trichloromethyl)carbonate.

“Carboxyl” refers to a —C(O)OH group.

“Alkoxycarbonyl” refers to a —C(O)O(alkyl) or (cycloalkyl) group,wherein the alkyl and cycloalkyl are defined as above.

“Optional” or “optionally” means that the event or circumstancedescribed subsequently can, but not necessary occur. Such expressionmeans the case that the event or circumstance does or does not occur.For example, “the heterocyclic group optionally substituted by an alkyl”means that an alkyl group can be, but is not necessarily present, andsuch expression includes the case that the heterocyclic group issubstituted with an alkyl and the heterocyclic group is not substitutedwith an alkyl.

“Substituted” refers to one or more hydrogen atoms in a group,preferably up to 5, more preferably 1 to 3 hydrogen atoms, eachindependently replaced with a corresponding number of substituents. Itgoes without saying that the substituents exist in their only possiblechemical position. The person skilled in the art is able to determinewhether the substitution is possible or impossible without payingexcessive efforts by experiment or theory. For example, an amino orhydroxy group having a free hydrogen bound to carbon atoms havingunsaturated bonds (such as olefinic) may be unstable.

A “pharmaceutical composition” refers to a mixture of one or more of thecompounds described in the present invention orphysiologically/pharmaceutically acceptable salts or prodrugs thereof,and other chemical components such as physiologically/pharmaceuticallyacceptable carriers and excipients. The purpose of a pharmaceuticalcomposition is to facilitate administration of a compound to anorganism, which is conducive to the absorption of the active ingredient,thus displaying biological activity.

R⁶˜R⁵ are as defined in formula (I).

Synthesis Method of the Present Compound

In order to achieve the object of the present invention, the presentinvention applies the following technical solution:

A process for preparing a compound of formula (I), or a tautomer,mesomer, racemate, enantiomer, or diastereomer thereof, or a mixturethereof, or a pharmaceutically acceptable salt thereof, comprises thefollowing steps:

a compound of formula (Ia) is reacted with R²X in the presence of analkaline reagent to obtain a compound of formula (Ib); the compound offormula (Ib) is subjected to a halogenating reaction under a catalyst toobtain a compound of formula (IA), wherein the catalyst is preferablyazobisisobutyronitrile, and the halogenating agent is preferablyN-bromosuccinimide; the compound of formula (IA) is reacted with theamine of NH(R⁴)(CH₂)nR⁵ to obtain a compound of formula (I);wherein: X is halogen; n, D, E, G, and R¹ to R⁵ are as defined informula (I);R³ is preferably an aryl, more preferably a phenyl, wherein the aryl isoptionally further substituted with one or more groups selected from thegroup consisting of —NHC(O)NHR⁶ and —NHC(O)NHOR⁶.

A process for preparing a compound of formula (I), or a tautomer,mesomer, racemate, enantiomer, or diastereomer thereof, or a mixturethereof, or a pharmaceutically acceptable salt thereof, comprises thefollowing steps:

the compound of formula (Ic) is subject to a halogenating reaction undera catalyst to obtain a compound of formula (Id), wherein the catalyst ispreferably azobisisobutyronitrile, and the halogenating agent ispreferably N-bromosuccinimide; the compound of formula (Id) is reactedwith the amine NH(R⁴)(CH₂)nR⁵ to obtain a compound of formula (Ie); thecompound of formula (Ie) is further heated under trifluoroacetic acid toobtain a compound of formula (If); the compound of formula (If) isreacted with triphosgene and an amine of R¹NH₂ successively to obtain acompound of formula (Ig); the compound of formula (Ig) is subject to acyclization reaction under an alkaline condition to obtain a compound offormula (IB); the compound of formula (IB) is reacted with R²X in thepresence of an alkaline reagent to obtain a compound of formula (I);wherein:X is halogen; Rb is alkyl;n, D, E, G, and R¹ to R⁵ are as defined in formula (I); andR³ is preferably an aryl, more preferably a phenyl, wherein the aryl isoptionally further substituted with one or more groups selected from thegroup consisting of —NHC(O)NHR⁶ and —NHC(O)NHOR⁶.

A process for preparing a compound of formula (II), or a tautomer,mesomer, racemate, enantiomer, or diastereomer thereof, or mixturethereof, or a pharmaceutically acceptable salt thereof, comprises thefollowing steps:

the compound of formula (IIa) is heated under trifluoroacetic acid toobtain a compound of formula (IIb); the compound of formula (IIb) isreacted with triphosgene and an amine of R¹NH₂ successively to obtain acompound of formula (IIc); the compound of formula (IIc) is subject to acyclization reaction under an alkaline to obtain a compound of formula(IId); the compound of formula (IId) is reacted with R²X in the presenceof an alkaline reagent to obtain a compound of formula (IIe); thecompound of formula (IIe) is subject to a halogenating reaction under acatalyst to obtain a compound of formula (IIA), wherein the catalyst ispreferably azobisisobutyronitrile, and the halogenating agent ispreferably N-bromosuccinimide; the compound of formula (IIA) is reactedwith an amine of NH(R⁴)(CH₂)nR⁵ to obtain a compound of formula (II);wherein: X is halogen; R^(b) is alkyl;n, and R¹ to R⁵ are as defined in formula (I);R³ is preferably an aryl, more preferably a phenyl, wherein the aryl isoptionally further substituted with one or more groups selected from thegroup consisting of —NHC(O)NHR⁶ and —NHC(O)NHOR⁶.

A process for preparing a compound of formula (III), or a tautomer,mesomer, racemate, enantiomer, or diastereomer thereof, or mixturethereof, or a pharmaceutically acceptable salt thereof, comprises thefollowing steps:

the compound of formula (IIIa) is reacted with triphosgene and an amineof R¹NH₂ successively to obtain a compound of formula (IIIb); thecompound of formula (IIIb) is reacted under an alkaline condition toobtain a compound of formula (IIIc); the compound of formula (IIIc) isreacted with R²X in the presence of an alkaline reagent to obtain acompound of formula (IIId); the compound of formula (IIId) is subject toa halogenating reaction under a catalyst to obtain a compound of formula(IIIA), wherein the catalyst is preferably azobisisobutyronitrile, andthe halogenating agent is preferably N-bromosuccinimide; the compound offormula (IIIA) is reacted with an amine of NH(R⁴)(CH₂)nR⁵ to obtain acompound of formula (III);wherein: X is halogen; R^(b) is alkyl;n, and R¹ to R⁵ are as defined in formula (I);R³ is preferably an aryl, more preferably a phenyl, wherein the aryl isoptionally further substituted with one or more groups selected from thegroup consisting of —NHC(O)NHR⁶ and —NHC(O)NHOR⁶.

A process for preparing a compound of formula (IV), or a tautomer,mesomer, racemate, enantiomer, or diastereomer thereof, or mixturethereof, or a pharmaceutically acceptable salt thereof, comprises thefollowing steps:

formula (IVa) is prepared according to schemes 1-4; the compound offormula (IVa) is reacted with R²X in the presence of an alkaline reagentto obtain a compound of formula (IVb); the compound of formula (IVb) issubject to a halogenating reaction under a catalyst to obtain a compoundof formula (IVc), wherein the catalyst is preferablyazobisisobutyronitrile, and the halogenating agent is preferablyN-bromosuccinimide; the compound of formula (IVc) is reacted with anamine of NH(R⁴)(CH₂)nR⁵ to obtain a compound of formula (IVd); thecompound of formula (IVd) is subject to a reduction reaction with ironin the presence of ammonium chloride to reduce a nitro group to an aminogroup to obtain a compound of formula (IVA); the compound of formula(IVA) is further reacted with an acylating reagent to obtain a compoundof formula (IV);wherein: X is halogen, and n, D, E, G. R^(a), and R¹ to R⁵ are asdefined in formula (I).

In the aforesaid technical solutions, the alkaline condition is providedby an organic base or an inorganic base, wherein the organic baseincludes, but is not limited to, triethylamine, pyridine, 2,6-lutidine,sodium methoxide, lithium hexamethyldisilazide, sodiumhexamethyldisilazide, n-butyllithium, potassium tert-butoxide ortetrabutyl ammonium bromide; and the inorganic base includes, but is notlimited to, sodium hydride, sodium carbonate, sodium bicarbonate,potassium carbonate, potassium bicarbonate, cesium carbonate, lithiumhydroxide, sodium hydroxide or potassium hydroxide. In the method of thepresent invention, the alkaline condition of cyclization is preferablyan organic base, and more preferably is sodium methoxide.

Reducing agent includes, but is not limited to, hydrogen and ironpowder.

Acylating agent includes, but is not limited to, carboxylic acid, acylchloride, sulfuryl chloride, halogenated methyl formate, isocyanate,triphosgene, and methoxyamine.

The invention will be further illustrated with reference to thefollowing specific examples. It should be understood that these examplesare merely intended to demonstrate the invention without limiting thescope of the invention.

The experimental methods in the following examples for which no specificconditions are indicated will be carried out according to conventionalconditions or recommended conditions of the raw materials and theproduct manufacturer. The experimental reagents for which no specificsources are indicated will be conventional reagents which arecommercially available.

EXAMPLES

Compound structures are identified by nuclear magnetic resonance (NMR)and/or mass spectrometry (MS). NMR chemical shift (δ) is given in 10⁻⁶(ppm). NMR is determined by Bruker AVANCE-400. The solvents for NMR aredeuterated-dimethyl sulfoxide (DMSO-d₆), deuterated-chloroform (CDCl₃)and deuterated-methanol (CD₃OD), with tetramethylsilane (TMS) as aninternal standard.

MS is determined by a FINNIGAN LCQAd (ESI) mass spectrometer(manufacturer: Thermo, type: Finnigan LCQ advantage MAX).

High performance liquid chromatography (HPLC) is determined on anAgilent 1200DAD high pressure liquid chromatography spectrometer(Sunfire C18 150×4.6 mm chromatographic column) and a Waters 2695-2996high pressure liquid chromatography spectrometer (Gimini C18 150×4.6 mmchromatographic column).

The average inhibition rate of kinase and IC₅₀ are determined by aNovoStar ELISA (BMG Co., Germany).

Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate is used forthin-layer silica gel chromatography (TLC). The dimension of the platesused in TLC was 0.15 mm to 0.2 mm, and the dimension of the plates usedin product purification is 0.4 mm to 0.5 mm.

Yantai Huanghai 200 to 300 mesh silica gel is used as carrier in columnchromatography.

The known starting materials of the present invention can be prepared byconventional synthesis methods in the art, or can be purchased from ABCRGmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBioInc., or Dari Chemical Company, etc.

Unless otherwise stated, the reactions can be carried out under nitrogenatmosphere or argon atmosphere.

The term “argon atmosphere” or “nitrogen atmosphere” means that areaction flask is equipped with a 1 L argon or nitrogen balloon.

Unless otherwise stated, the solution in the examples refers to anaqueous solution.

Unless otherwise stated, the reaction temperature in the examples isroom temperature, and the range of the room temperature was 20° C. to30° C.

The reaction process is monitored by thin layer chromatography (TLC),and the system of developing solvent includes: A: dichloromethane andmethanol system, B: n-hexane and ethyl acetate system, C: petroleumether and ethyl acetate system, D: acetone. The ratio of the solvent canbe adjusted according to the polarity of the compounds.

The elution system for purification of the compounds by columnchromatography and thin layer chromatography includes: A:dichloromethane and methanol system, B: n-hexane and ethyl acetatesystem, C: dichloromethane and acetone system, D: ethyl acetate anddichloromethane system, E: ethyl acetate and dichloromethane andn-hexane system, F: ethyl acetate and dichloromethane and acetonesystem. The volume ratio of the solvent can be adjusted according to thepolarity of the compounds, and sometimes a little alkaline reagent, suchas triethylamine, or acidic reagent, such as acetic acid, may also beadded.

Example 1N-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)acetamide

Step 1 Ethyl3-amino-5-methyl-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate

Ethyl3-(tert-butylamino)-5-methyl-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate1a (12.10 g, 34.93 mmol, prepared by a well known method disclosed in“Synthesis, 1988, (3), 203-207”) and 100 mL of trifluoroacetic acid wereadded to a 250 mL of eggplant shaped flask successively. The mixture wasstirred for 2.5 hours at 85° C., and then the reaction was stopped. Thereaction solution was cooled to room temperature, and concentrated underreduced pressure. The residue was added dropwise with saturated sodiumbicarbonate solution to adjust the pH>7, and filtrated. The filter cakewas dissolved in dichloromethane and the organic phase was dried overanhydrous sodium sulfate, and filtered. The filtrate was concentratedunder reduced pressure and the residue was slurried with 50 mL of amixture of dichloromethane and methanol (V/V=20:1), and filtered. Theproduct was dried to obtain 5.30 g of yellow solid. The mother solutionwas concentrated under reduced pressure and the residue was purified bysilica gel column chromatography with elution system A to obtain thetitle compound ethyl3-amino-5-methyl-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate 1b (totalproduct 6.07 g, yellow solid), yield: 60.1%.

MS m/z (ESI): 291.2 [M+1]

Step 2 Ethyl3-(3-(2-fluoro-3-methoxyphenyl)ureido)-5-methyl-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate

Ethyl 3-amino-5-methyl-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate 1b(550 mg, 1.89 mmol) was dissolved in 50 mL of dichloromethane. Themixture, which was insufficiently soluble at room temperature, was addedwith triethylamine (0.66 mL, 4.75 mmol) and triphosgene (225 mg, 0.76mmol), and stirred until it was dissolved completely. After stirring for30 minutes, the reaction solution was added with2-fluoro-3-methoxyaniline (283 mg, 2 mmol) and stirred for 12 hours atroom temperature, then the reaction was stopped. The reaction solutionwas concentrated under reduced pressure, and the residues was added with20 mL of water, and extracted with dichloromethane (50 mL×2). Theorganic phases were combined, dried over anhydrous sodium sulfate, andfiltered. The filtrate was concentrated under reduced pressure and theresidue was purified by silica gel column chromatography with elutionsystem A to obtain the title compound ethyl3-(3-(2-fluoro-3-methoxyphenyl)ureido)-5-methyl-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate1c (640 mg, white solid), yield: 73.9%.

MS m/z (ESI): 458.1 [M+1]

Step 35-(2-fluoro-3-methoxyphenyl)-3-methyl-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

Ethyl3-(3-(2-fluoro-3-methoxyphenyl)ureido)-5-methyl-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate1c (640 mg, 1.40 mmol) was dissolved in 30 mL of ethanol, and then addedwith sodium methoxide (190 mg, 3.52 mmol). After reacting for 3 hours atroom temperature, the reaction solution was heated to 80° C. and furtherreacted for 1 hour. Then, the reaction was stopped. The reactionsolution was cooled to room temperature, and then added with 3.5 mL of 1M hydrochloric acid solution, and filtered. The filter cake was washedwith water (20 mL×1), ethanol (10 mL×1), and methyl tert-butyl ether (5mL×1) successively, and dried to obtain the title compound5-(2-fluoro-3-methoxyphenyl)-3-methyl-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione1d (536 mg, light yellow solid), yield: 93.2%.

MS m/z (ESI): 412.2 [M+1]

Step 47-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)-3-methyl-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

5-(2-fluoro-3-methoxyphenyl)-3-methyl-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione1d (530 mg, 1.29 mmol), 2-(chloromethyl)-1,3-difluorobenzene (230 mg,1.41 mmol), potassium iodide (235 mg, 1.41 mmol), and potassiumcarbonate (196 mg, 1.42 mmol) were dissolved in 15 mL of N,N-dimethylformamide, successively. After reacting for 24 hours at roomtemperature, the reaction was stopped, and the reaction solution wasconcentrated under reduced pressure. The residue was added with 30 mL ofwater and 20 mL of methyl tert-butyl ether under stirring, and thenfiltered. The filter cake was dissolved in 50 mL of dichloromethane,dried over anhydrous sodium sulfate, and filtered. The filtrate wasconcentrated under reduced pressure to obtain 570 mg of solid. Themother solution was extracted, the organic phase was washed with water(20 mL×3), and saturated sodium chloride solution (20 mL×1),successively, dried over anhydrous sodium sulfate, and filtered. Thefiltrate was concentrated under reduced pressure and the residue wasslurried with 15 mL of a mixture of n-hexane and methyl tert-butyl ether(V/V=1:1), and filtered. The filter cake was dried to obtain 110 mg ofsolid. The title compound7-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)-3-methyl-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione1e (total product 680 mg, tan solid) was obtained, yield: 98.3%.

MS m/z (ESI): 538.2 [M+1]

Step 53-(bromomethyl)-7-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

Under argon atmosphere,7-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)-3-methyl-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione1e (660 mg, 1.23 mmol), azobisisobutyronitrile (24 mg, 0.15 mmol), andN-bromosuccinimide (262 mg, 1.47 mmol) were dissolved in 20 mL ofchlorobenzene, successively. After reacting for 12 hours at 85° C., thereaction was stopped and the reaction solution was cooled to roomtemperature, and then washed with saturated sodium chloride solution (15mL×1) and saturated sodium thiosulfate solution (15 mL×1), successively.The aqueous phase was extracted with dichloromethane (40 mL×2). Theorganic phases were combined, dried over anhydrous sodium sulfate, andfiltered. The filtrate was concentrated under reduced pressure to obtainthe crude title compound3-(bromomethyl)-7-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione1f (900 mg, light yellow solid), which was used directly in the nextstep.

MS m/z (ESI): 616.1 [M+1]

Step 67-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

The crude3-(bromomethyl)-7-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione1f (900 mg, 1.46 mmol) was dissolved in a 3 mL solution of dimethylaminein tetrahydrofuran. After reacting for 12 hours at room temperature, thereaction was stopped and the reaction solution was concentrated underreduced pressure. The residue was purified via silica gel columnchromatography with eluent system D to obtain 627 mg of crude compound.The crude compound was purified by thin-layer chromatography with eluentsystem E to obtain the title compound7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione1g (223 mg, light yellow solid), yield: 31.0%.

MS m/z (ESI): 581.3 [M+1]

Step 72-(4-aminophenyl)-7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione1g (212 mg, 0.36 mmol) was dissolved in 8 mL of formic acid, and thenadded with a 0.25 mL solution of 4 M HCl in 1, 4-dioxane and 10%palladium on carbon (40 mg). The mixture was purged with hydrogen threetimes and reacted for 2 hours under hydrogen atmosphere and normalpressure. Then the reaction was stopped, and the reaction mixture wasfiltered. The filter cake was washed with methanol (20 mL×2). Theorganic phase was concentrated under reduced pressure. The residue wasadded with 50 mL of dichloromethane and 15 mL of saturated sodiumbicarbonate solution, and stirred for 10 minutes. The saturated sodiumbicarbonate solution was added dropwise to adjust the pH>7, and theaqueous phase was subject to extraction. The aqueous phase was extractedwith dichloromethane (20 mL×3). The organic phases were combined, driedover anhydrous sodium sulfate, and filtered. The filtrate wasconcentrated under reduced pressure, and the residue was purified bythin-layer chromatography with eluent system A to obtain the titlecompound2-(4-aminophenyl)-7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione1h (180 mg, white solid), yield: 89.5%.

MS m/z (ESI): 551.3 [M+1]

Step 8N-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)acetamide

2-(4-aminophenyl)-7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione1h (33 mg, 0.059 mmol) was dissolved in 5 mL of dichloromethane, andadded with triethylamine (13 μL, 0.094 mmol) and acetic anhydride (7 μL,0.072 mmol). After reacting for 12 hours at room temperature, thereaction was incomplete. 5 μL of acetic anhydride was then supplemented,and the mixture was heated to 35° C. and further reacted for 2 hours.The reaction was stopped and the reaction solution was added with 2 mLof saturated sodium bicarbonate solution, stirred for 10 minutes, andextracted. The aqueous phase was extracted with dichloromethane (15mL×3). The organic phases were combined, dried over anhydrous sodiumsulfate, and filtered. The filtrate was concentrated under reducedpressure, and the residue was purified by thin layer chromatography withelution system A to obtain the title compoundN-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)acetamide1 (25 mg, white solid), yield: 71.4%.

MS m/z (ESI): 593.1 [M+1]

¹H NMR (400 MHz, CDCl₃) δ 7.89 (d, 2H), 7.67 (d, 2H), 7.36 (s, 1H),7.25-7.30 (m, 1H), 7.19 (t, 1H), 7.08 (t, 1H), 7.89-7.94 (m, 3H), 5.45(s, 2H), 3.93 (s, 3H), 3.69-3.70 (m, 2H), 2.35 (s, 6H), 2.25 (s, 3H).

Example 21-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxyurea

Step 11-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxyurea

2-(4-aminophenyl)-7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione1h (30 mg, 0.054 mmol) was dissolved in 5 mL of dichloromethane, andadded with triethylamine (0.045 mL, 0.32 mmol) and triphosgene (6.5 mg,0.022 mmol). After reacting for 1 hour, the reaction solution was addedwith methoxyamine (6.8 mg, 0.081 mmol), and further reacted for 12 hoursat room temperature, and then supplemented with triphosgene (18 mg,0.061 mmol), and further reacted for 1 hour at 35° C., followed byaddition of methoxyamine (100 mg, 1.20 mmol) and continued reaction for2 hours at 35° C. The reaction was stopped, and the reaction solutionwas added with 10 mL of saturated sodium bicarbonate solution, andextracted. The aqueous phase was extracted with dichloromethane (15mL×3). The organic phases were combined, dried over anhydrous sodiumsulfate, and filtered. The filtrate was concentrated under reducedpressure and the residue was purified by thin layer chromatography withelution system A to obtain the title compound1-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxyurea2 (12 mg, pale yellow solid), yield: 35.3%.

MS m/z (ESI): 624.5 [M+1]

¹H NMR (400 MHz, CDCl₃) δ 7.90 (d, 2H), 7.71 (s, 1H), 7.67 (d, 2H),7.20-7.30 (m, 3H), 7.06-7.08 (m, 1H), 6.89-6.94 (m, 3H), 5.45 (s, 2H),3.94 (s, 3H), 3.87 (s, 3H), 3.86 (s, 2H), 2.35 (s, 6H).

Example 31-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-ethylurea

Step 11-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-ethylurea

2-(4-aminophenyl)-7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione1h (26 mg, 0.047 mmol) was dissolved in 3 mL of tetrahydrofuran, addedwith ethyl isocyanate (55 μL, 0.69 mmol), and reacted for 12 hours at35° C. The reaction was stopped, and the reaction solution wasconcentrated under reduced pressure. The residue was added with 10 mL ofwater and 20 mL of dichloromethane, and extracted. The aqueous phase wasextracted with dichloromethane (15 mL×3). The organic phases werecombined, dried over anhydrous sodium sulfate, and filtered. Thefiltrate was concentrated under reduced pressure and the residue waspurified by thin layer chromatography with elution system A to obtainthe title compound1-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-ethylurea3 (12 mg, white solid), yield: 41.4%.

MS m/z (ESI): 622.5 [M+1]

¹H NMR (400 MHz, CDCl₃) δ 7.80 (d, 2H), 7.45 (d, 2H), 7.22-7.26 (m, 2H),7.13-7.15 (m, 1H), 6.99-7.04 (m, 1H), 6.84-6.89 (m, 3H), 6.45 (s, 1H),5.40 (s, 2H), 3.89 (s, 3H), 3.65 (s, 2H), 3.33-3.35 (m, 2H), 2.30 (s,6H), 1.20 (t, 3H).

Example 4 Methyl(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)carbamate

Step 1 Methyl(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)carbamate

2-(4-aminophenyl)-7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione1h (35 mg, 0.064 mmol) was dissolved in 5 mL of dichloromethane, andadded with triethylamine (0.8 mL, 5.8 mmol) and methyl chloroformate(0.5 mL, 4.14 mmol). After reacting for 12 hours at 35° C., the reactionwas stopped and cooled to room temperature. The reaction solution wasadded with 10 mL of saturated sodium bicarbonate solution, andextracted.

The aqueous phase was extracted with dichloromethane (15 mL×3). Theorganic phases were combined, dried over anhydrous sodium sulfate, andfiltered. The filtrate was concentrated under reduced pressure, and theresidue was purified by thin layer chromatography with elution system Ato obtain the title compound methyl(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)carbamate4 (24 mg, white solid), yield: 61.5%.

MS m/z (ESI): 609.5 [M+1]

¹H NMR (400 MHz, CDCl₃) δ 7.88 (d, 2H), 7.55 (d, 2H), 7.25-7.27 (m, 1H),7.18-7.20 (m, 1H), 7.06-7.08 (m, 1H), 6.88-6.94 (m, 3H), 6.78 (s, 1H),5.45 (s, 2H), 3.93 (s, 3H), 3.85 (s, 3H), 3.65-3.73 (m, 2H), 2.35 (s,6H).

Example 51-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methylurea

Step 11-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methylurea

Acetic acid (46 mg, 0.77 mmol) was dissolved in 1 mL of toluene andadded with N,N-diisopropylethylamine (0.2 mL, 1.13 mmol) anddiphenylphosphoryl azide (0.16 mL, 0.73 mmol). The mixture was stirredfor 1 hour at 70° C., and then cooled in an ice bath, followed byaddition of2-(4-aminophenyl)-7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione1h (30 mg, 0.050 mmol) and 2 mL dichloromethane in an ice bath. Themixture was reacted for 12 hours at room temperature. The reaction wasstopped, and the reaction solution was added with 10 mL of saturatedsodium bicarbonate solution, and extracted with dichloromethane (25mL×1). The organic phase was washed with water (10 mL×1) and saturatedsodium chloride solution (10 mL×1), dried over anhydrous sodium sulfate,and filtered. The filtrate was concentrated under reduced pressure, andthe residues were purified by thin layer chromatography with elutionsystem A to obtain the title compound1-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methylurea5 (8 mg, white solid), yield: 24.4%.

MS m/z (ESI): 607.9 [M+1]

¹H NMR (400 MHz, CDCl₃) δ 7.79 (d, 2H), 7.50 (d, 2H), 7.27-7.30 (m, 1H),7.19 (t, 1H), 7.06 (t, 1H), 6.880-6.93 (m, 4H), 5.44 (s, 2H), 4.95 (s,1H), 3.92 (s, 3H), 3.78 (s, 2H), 2.88 (d, 3H), 2.39 (s, 6H).

Example 61-(4-(3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-ethylurea

Step 15-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-3-methyl-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

5-(2-fluoro-3-methoxyphenyl)-3-methyl-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione1d (1.90 g, 4.62 mmol),2-(bromomethyl)-1-fluoro-3-(trifluoromethyl)benzene (1.31 g, 5.10 mmol),potassium iodide (844 mg, 5.08 mmol), and potassium carbonate (702 mg,5.09 mmol) were dissolved in 54 mL of N,N-dimethylformamidesuccessively. After stirring for 20 hours at room temperature, thereaction was stopped, and the reaction solution was concentrated underreduced pressure. The residues were added with water and ether to slurryfor 1 hour, and filtered. The filter cake was washed with ether (10mL×3) and dried to obtain the title compound5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-3-methyl-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione6a (1.91 g, white solid), yield 70.7%.

MS m/z (ESI): 588.1 [M+1]

Step 23-(bromomethyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-3-methyl-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione6a (1.81 g, 3.08 mmol), azobisisobutyronitrile (61 mg, 0.37 mmol) andN-bromosuccinimide (660 mg, 3.71 mmol) were dissolved in 53 mL ofchlorobenzene. The resulting solution was purged with argon three times,and reacted for 19 hours at 85° C. under argon atmosphere. The reactionwas stopped, and the reaction solution was washed with saturated sodiumthiosulfate solution (30 mL×1) and saturated sodium chloride solution(50 mL×2), dried over anhydrous sodium sulfate, and filtered. Thefiltrate was concentrated under reduced pressure to obtain the crudetitle compound3-(bromomethyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione6b (2.18 g, pale yellow solid), which was used directly in the nextstep.

MS m/z (ESI): 666.0 [M+1]

Step 33-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

The crude3-(bromomethyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione6b (2.18 g, 3.27 mmol) and dimethylamine (2 M solution in THF) (6.8 mL13.6 mmol) were added to the reaction flask and stirred for 12 hours atroom temperature. The reaction was stopped, and the reaction solutionwas concentrated under reduced pressure. The residue was purified bypreparative separation to obtain the title compound3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione6c (455 mg, white solid), yield: 22.1%.

MS m/z (ESI): 630.8 [M+1]

Step 42-(4-aminophenyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione6c (200 mg, 0.32 mmol) was dissolved in 8 mL of formic acid, and addedwith 0.25 mL solution of 4 M HCl in 1,4-dioxane and 10% of Pd/C (50 mg).The mixture was purged with hydrogen three times, and stirred for 2hours at room temperature. The reaction was stopped and filtered. Thefilter cake was washed with a small amount of methanol. The filtrate wasconcentrated under reduced pressure, added with 50 mL of dichloromethaneand 20 mL of sodium bicarbonate solution, and stirred for 10 minutes.The aqueous phase was adjusted to pH 8, and extracted. The aqueous phasewas extracted with dichloromethane (25 mL×2) and the organic phases werecombined, dried over anhydrous sodium sulfate, and filtered. Thefiltrate was concentrated under reduced pressure and the residue waspurified by thin layer chromatography with elution system A to obtainthe title compound2-(4-aminophenyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione6d (135 mg, white solid), yield: 71.1%.

MS m/z (ESI): 600.9 [M+1]

Step 51-(4-(3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-ethylurea

2-(4-aminophenyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione6d (30 mg, 0.050 mmol) was dissolved in 1 mL of dichloromethane, andadded with ethyl isocyanate (0.06 mL, 0.76 mmol). After reacting for 12hours at 35° C., the reaction was stopped and the reaction solution wasadded with 5 mL of saturated sodium bicarbonate solution and 20 mL ofdichloromethane solution, and subjected to extraction. The organic phasewas washed with water (10 mL×1) and saturated sodium chloride solution(5 mL×1), dried over anhydrous sodium sulfate, and filtered, Thefiltrate was concentrated under reduced pressure, and the residue waspurified by thin layer chromatography with elution system A to obtainthe title compound1-(4-(3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-ethylurea6 (15 mg, white solid), yield: 44.1%.

MS m/z (ESI): 671.9 [M+1]

¹H NMR (400 MHz, CDCl₃) δ 7.75 (d, 2H), 7.51 (d, 1H), 7.46 (d, 2H),7.37-7.40 (m, 1H), 7.18-7.24 (m, 2H), 7.15 (t, 1H), 6.91 (t, 1H), 6.85(s, 1H), 5.60 (s, 2H), 4.92 (s, 1H), 3.90 (s, 3H), 3.69-3.76 (m, 2H),3.28-3.35 (m, 2H), 2.35 (s, 6H), 1.19 (t, 3H).

Example 71-(4-(3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxyurea

Step 1S-(4-(3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxyurea

2-(4-aminophenyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione6d (30 mg, 0.050 mmol) was dissolved in 3 mL of dichloromethane, andadded with triethylamine (0.07 mL, 0.50 mmol) and triphosgene (15 mg,0.051 mmol). After reacting for 1 hour at room temperature, the reactionwas stopped and the reaction solution was added with 10 mL of water, andthen extracted with dichloromethane (25 mL×1). The organic phase waswashed with saturated sodium bicarbonate solution (10 mL×1), water (10mL×1), and saturated sodium chloride solution (10 mL×1), successively,dried over anhydrous sodium sulfate, and filtered. The filtrate wasconcentrated under reduced pressure and the residue was purified by thinlayer chromatography with elution system A to obtain the title compound1-(4-(3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxyurea7 (12 mg, white solid), yield: 33.6%.

MS m/z (ESI): 673.8 [M+1]

¹H NMR (400 MHz, CDCl₃) δ 7.85 (d, 2H), 7.64-7.70 (m, 3H), 7.53 (d, 1H),7.39-7.42 (m, 1H), 7.23-7.28 (m, 3H), 7.06 (t, 1H), 6.92 (t, 1H),5.57-5.65 (m, 2H), 3.93 (s, 3H), 3.87 (s, 3H), 3.72 (s, 2H), 2.36 (s,6H).

Example 81-(4-(3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methylurea

Step 11-(4-(3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methylurea

Acetic acid (62 mg, 1.03 mmol) was dissolved in 1 mL of toluene, andadded with N,N-diisopropylethylamine (0.28 mL, 1.58 mmol) anddiphenylphosphoryl azide (0.20 mL, 0.98 mmol). After reacting for 1.5hours at 70° C., the reaction solution was added with2-(4-aminophenyl)-3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione6d (30 mg, 0.050 mmol) and 1 mL of dichloromethane, and further reactedfor 12 hours at room temperature. The reaction was stopped. The reactionsolution was added with 5 mL of saturated sodium bicarbonate, and thenextracted with dichloromethane (25 mL×1). The organic phase was washedwith water (10 mL×1) and saturated sodium chloride solution (10 mL×1),dried over anhydrous sodium sulfate, and filtered. The filtrate wasconcentrated under reduced pressure and the residue was purified by thinlayer chromatography with elution system A to obtain the crude titlecompound, which was further purified by thin layer chromatography withelution system A to obtain the title compound1-(4-(3-((dimethylamino)methyl)-5-(2-fluoro-3-methoxyphenyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methylurea8 (13 mg, white solid), yield 39.4%.

MS m/z (ESI): 658.2 [M+1]

¹H NMR (400 MHz, CDCl₃) δ 7.76 (d, 2H), 7.51-7.53 (m, 1H), 7.47 (d, 2H),7.39-7.40 (m, 1H), 7.24 (t, 1H), 7.19 (t, 1H), 7.04 (t, 1H), 6.89 (t,1H), 6.74 (m, 1H), 5.56-5.64 (m, 2H), 4.85 (s, 1H), 3.92 (s, 3H), 3.77(s, 2H), 2.88 (d, 3H), 2.38 (s, 6H).

Example 91-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methylurea

Step 1 Ethyl5-(bromomethyl)-3-(tert-butylamino)-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate

Ethyl3-(tert-butylamino)-5-methyl-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate1a (23 g, 66.40 mmol), azobisisobutyronitrile (2.18 g, 13.28 mmol),N-bromosuccinimide (14.18 g, 79.68 mmol), and 400 mL of chlorobenzenewere added to a 1 L single-port reaction flask. The reaction solutionwas stirred uniformly and purged with argon three times. After reactingfor 64 hours at 85° C., the reaction was stopped. The reaction solutionwas concentrated under reduced pressure. The residues were added with300 mL of dichloromethane, and washed with water (100 mL×2), saturatedsodium thiosulfate solution (100 mL×2), and saturated sodium chloridesolution (100 mL×2), successively. The organic phase was dried overanhydrous sodium sulfate, filtered, and the filtrate was concentratedunder reduced pressure to obtain the crude title compound ethyl5-(bromomethyl)-3-(tert-butylamino)-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate9a (25 g, brown oil), which was used directly in the next step.

MS m/z (ESI): 427.1 [M+2]

Step 2 Ethyl3-(tert-butylamino)-5-((dimethylamino)methyl)-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate

The crude ethyl5-(bromomethyl)-3-(tert-butylamino)-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate9a (25 g, 58.78 mmol), a solution of dimethyl amine in THF (88 mL,176.35 mmol) and 120 mL of THF were added to a 500 mL single-portreaction flask, and stirred until dissolution. After stirring for 3hours at room temperature, the reaction was stopped. The reactionsolution was concentrated under reduced pressure. The residue waspurified by silica gel column chromatography with eluent system C toobtain the title compound ethyl3-(tert-butylamino)-5-((dimethylamino)methyl)-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate9b (6 g, brown oil), yield 26.2%.

MS m/z (ESI): 390.2 [M+1]

Step 3 Methyl3-amino-5-((dimethylamino)methyl)-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate

Ethyl3-(tert-butylamino)-5-((dimethylamino)methyl)-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate9b (6 g, 15.41 mmol) and 80 mL of trifluoroacetic acid were added to a100 mL single-port reaction flask and stirred uniformly. The mixture washeated to reflux for 1 hour and the reaction was stopped. The reactionsolution was concentrated under reduced pressure. The residue was addedwith 50 mL of dichloromethane, and then added dropwise with saturatedsodium bicarbonate solution to adjust the pH >7. The organic phase wasdried over anhydrous magnesium sulfate, filtered, and concentrated underreduced pressure. The residue was added with 100 mL of methanol and 20mL of dichloromethane, and stirred to dissolution. The resultingsolution was added with potassium carbonate (6.37 g, 46.16 mmol), andstirred for 17 hours at room temperature, and then filtered. Thefiltrate was concentrated under reduced pressure and the residue waspurified by silica gel column chromatography with eluent system B toobtain the title compound methyl3-amino-5-((dimethylamino)methyl)-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate9c (3.30 g, yellow solid), yield 67.1%.

MS m/z (ESI): 320.2 [M+1]

Step 4 Methyl5-((dimethylamino)methyl)-3-(3-(6-methoxypyridazin-3-yl)ureido)-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate

Methyl3-amino-5-((dimethylamino)methyl)-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate9c (3.30 g, 10.33 mmol) and 100 mL of dichloromethane were added to a250 mL single-port reaction flask, and stirred uniformly. The resultingsolution was added with triethylamine (5.8 mL, 41.32 mmol) andtriphosgene (1.84 g, 6.20 mmol), and then stirred for 1 hour at roomtemperature. The reaction solution was added with3-amino-6-methoxypyridazine (1.94 g, 15.50 mmol, prepared by a knownmethod disclosed in “Journal of Medicinal Chemistry, 2006, 49(14),4409-4424”), and stirred for 2 hours at room temperature. Then thereaction was stopped. The reaction solution was added with 200 mL ofwater, and extracted with dichloromethane (100 mL×3). The organic phasewas concentrated under reduced pressure, and the residue was purified bysilica gel column chromatography with eluent system B to obtain thetitle compound methyl5-((dimethylamino)methyl)-3-(3-(6-methoxypyridazin-3-yl)ureido)-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate9d (2.80 g, yellow solid), yield 57.6%.

MS m/z (ESI): 470.9 [M+1]

Step 53-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

Methyl5-((dimethylamino)methyl)-3-(3-(6-methoxypyridazin-3-yl)ureido)-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate9d (2.80 g, 5.95 mmol) and 120 mL of methanol were added to a 250 mLsingle-port reaction flask, and stirred uniformly. The resultingsolution was added with sodium methoxide (1.61 g, 29.76 mmol). Themixture was heated to 50° C. and stirred for 4 hours, and then thereaction was stopped. The reaction solution was concentrated underreduced pressure. The residue was added with 100 mL of water, and thenadded dropwise with concentrated hydrochloric acid to adjust the pH to 5to 6. The solution was extracted with dichloromethane (100 mL×3). Theorganic phase was concentrated under reduced pressure, and the residuewas purified by silica gel column chromatography with elution system Bto obtain the title compound3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione9e (1.80 g, yellow solid), yield 69.0%.

MS m/z (ESI): 438.9 [M+1]

Step 67-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione9e (900 mg, 2.05 mmol) and 50 mL of N,N-dimethylformamide were added toa 100 mL single-neck reactor flask, and stirred uniformly. The resultingsolution was added with 2,6-difluorobenzyl chloride (367 mg, 2.26 mmol)and potassium carbonate (850 mg, 6.16 mmol). The mixture was heated to40° C. and stirred for 17 hours, then the reaction was stopped. Thereaction solution was added with 250 mL of water, and stirred for 20minutes. The mixture was filtered, and the filter cake was dried invacuo to obtain the title compound7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione9f (1.10 g, yellow solid), yield 94.9%.

MS m/z (ESI): 564.9 [M+1]

Step 72-(4-aminophenyl)-7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione9f (1 g, 1.77 mmol), 10% palladium on carbon (200 mg), and 80 mL ofmethanol were added to a 250 mL single-port reaction flask, and stirreduniformly. The resulting solution was purged with hydrogen 6 times, andthen stirred for 17 hours at room temperature. The reaction was stopped.The reaction solution was filtered, and the filtrate was concentratedunder reduced pressure to obtain the title compound2-(4-aminophenyl)-7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione9g (785 mg, yellow solid), yield 82.9%.

MS m/z (ESI): 534.9 [M+1]

Step 8S-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methylurea

Acetic acid (483 mg, 8.04 mmol) and 15 mL of toluene were stirred todissolution in 600 mL autoclave. The resulting solution was added withN,N-diisopropylethylamine (2.1 mL, 12.07 mmol) and diphenylphosphorylazide (2.22 g, 8.07 mmol), successively. The mixture was heated to 75°C. and stirred for 1.5 hours. The reaction solution was added with 10 mLsolution of2-(4-aminophenyl)-7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione9g (480 mg, 0.80 mmol) in THF in an ice bath at 0° C. After completionof addition, the reaction solution was heated to 55° C. and stirred for16 hours. Then the reaction was stopped. The reaction solution was addedwith 20 mL of dichloromethane and 20 mL of water, and subject toextraction. The organic phase was washed with saturated sodiumbicarbonate solution (50 mL×1), water (50 mL×1), and saturated sodiumchloride solution (50 mL×1), successively. The organic phase was driedover anhydrous sodium sulfate, and filtered. The filtrate wasconcentrated under reduced pressure and the residue was purified bysilica gel column chromatography with eluent system A to obtain thecrude product, which was further purified by thin layer chromatographywith eluent system A to obtain the crude product (387 mg, brown solid),crude yield: 81.3%. The two crude batches were combined to obtain atotal product of 823 mg, which was purified by thin layer chromatographywith eluent system F. The resulting product was dissolved in methanol,and concentrated under reduced pressure until a solid was separated,which was subject to crystallization to obtain the title compound1-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methylurea9 (450 mg, white solid), total yield: 36.0%.

MS m/z (ESI): 592.3[M+1]

¹H NMR (400 MHz, CDCl₃) δ 8.17 (s, 1H), 7.54 (s, 1H), 7.42-7.45 (m, 4H),7.24-7.30 (m, 2H), 6.88 (t, 2H), 5.95 (s, 1H), 5.38 (s, 2H), 4.23 (s,3H), 3.69 (s, 2H), 2.91 (s, 3H), 2.33 (s, 6H).

Example 101-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-4,6-dioxo-4,5,67-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-ethylurea

Step 11-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-ethylurea

2-(4-aminophenyl)-7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione9g (22 mg, 0.041 mmol) was dissolved in 2 mL of dichloromethane, andadded with ethyl isocyanate (0.1 mL, 1.26 mmol). After reacting for 12hours at 35° C., the reaction was stopped. The reaction solution wasadded with 5 mL of saturated sodium bicarbonate solution, and extractedwith dichloromethane (30 mL×1). The organic phase was washed with water(10 mL×1), and saturated sodium chloride solution (10 mL×1),successively, dried over anhydrous sodium sulfate, and filtered. Thefiltrate was concentrated under reduced pressure, and the residue waspurified by thin layer chromatography with elution system A to obtainthe title compound1-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-ethylurea10 (10 mg, white solid), yield: 40.0%.

MS m/z (ESI): 606.2 [M+1]

¹H NMR (400 MHz, CDCl₃) δ 8.07 (s, 1H), 7.55 (d, 1H), 7.38-7.43 (m, 4H),7.25-7.28 (m, 2H), 6.858 (t, 2H), 5.87 (t, 1H), 5.38 (s, 2H), 4.23 (s,3H), 3.50 (s, 2H), 3.37 (t, 2H), 2.25 (s, 6H), 1.23 (t, 3H).

Example 1114-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxyurea

Step 11-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxyurea

2-(4-aminophenyl)-7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione9g (100 mg, 0.18 mmol) was dissolved in 5 mL of dichloromethane, andadded with N,N-diisopropylethylamine (0.5 mL, 2.90 mmol) and triphosgene(46 mg, 0.15 mmol). The mixture was reacted for 45 minutes at roomtemperature, and then added with methoxyamine hydrochloride (92 mg, 1.12mmol). The mixture was further reacted for 12 hours at 40° C., and thenthe reaction was stopped. The reaction solution was added with 5 mL ofsaturated sodium bicarbonate solution, and then extracted withdichloromethane (25 mL×1). The organic phase was washed with water (10mL×1) and saturated sodium chloride solution (10 mL×1), successively,dried over anhydrous sodium sulfate, and filtered. The filtrate wasconcentrated under reduced pressure, and the residue was purified bythin layer chromatography with elution system A to obtain 62 mg of crudeproduct, which was further purified by thin layer chromatography withelution system A for two times to obtain the title compound1-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxyurea11 (45 mg, white solid), yield: 24.7%.

MS m/z (ESI): 608.3 [M+1]

¹H NMR (400 MHz, CDCl₃) δ 7.90 (d, 2H), 7.73 (s, 1H), 7.68 (d, 2H), 7.41(d, 1H), 7.26-7.30 (m, 1H), 7.22 (s, 1H), 7.13 (s, 1H), 6.92 (t, 2H),5.44 (s, 2H), 4.21 (s, 3H), 3.88 (s, 3H), 3.70 (s, 2H), 2.35 (s, 6H).

Example 121-(4-(3-((dimethylamino)methyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-5-(6-methoxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methylurea

Step 1 Ethyl 3-(3-(6-methoxypyridazin-3-yl)ureido)-5-methyl-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate

Ethyl 3-amino-5-methyl-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate 1b(10.80 g, 37.20 mmol) was dissolved in 500 mL of dichloromethane, andthen added with triethylamine (20 mL, 144 mmol) and triphosgene (5.20 g,17.5 mmol), successively. After reacting for 1 hour at room temperature,the reaction solution was added with 3-amino-6-methoxypyridazine (6.60g, 52.3 mmol), and stirred for 12 hours at room temperature. Then thereaction was stopped. The resulting products were combined, and addedwith 100 mL of water. The solution was subjected to extraction. Theorganic phase was washed with water (100 mL×1) and saturated sodiumchloride solution (100 mL×1), dried over anhydrous sodium sulfate, andfiltered. The filtrate was concentrated under reduced pressure and theresidue was purified by silica gel column chromatography with eluentsystem C to obtain the title compound ethyl3-(3-(6-methoxypyridazin-3-yl)ureido)-5-methyl-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate12a (8.72 g, pale yellow solid), yield: 53.0%.

Step 25-(6-methoxypyridazin-3-yl)-3-methyl-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

Ethyl3-(3-(6-methoxypyridazin-3-yl)ureido)-5-methyl-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate12a (8.72 g, 19.70 mmol) was dissolved in 150 mL of methanol, and addedwith sodium methoxide (2.70 g, 50 mmol). The mixture was heated to 55°C. and stirred for 12 hours, then the reaction was stopped. The reactionsolution was concentrated under reduced pressure. The residue was addeddropwise with 1 M hydrochloric acid solution to adjust the pH<7, andfiltered. The filter cake was washed with water (30 mL×1), methanol (30mL×1), and ether (20 mL×1), successively, and dried in vacuo to obtainthe crude title compound5-(6-methoxypyridazin-3-yl)-3-methyl-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione12b (8.34 mg, light yellow solid), which was used directly in the nextstep.

MS m/z (ESI): 396.0 [M+1]

Step 37-(2-fluoro-6-(trifluoromethyl)benzyl)-5-(6-methoxypyridazin-3-yl)-3-methyl-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

2-(bromomethyl)-1-fluoro-3-(trifluoromethyl)benzene (purchased fromSinopharm Chemical Reagent Co., Ltd., product number XW239870821) (308mg, 1.20 mmol) was dissolved in 20 mL of N,N-dimethyl formamide, andadded with the crude5-(6-methoxypyridazin-3-yl)-3-methyl-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione12b (395 mg, 1 mmol) and potassium carbonate (276 mg, 2 mmol),successively. After reacting for 2 hours at room temperature, thereaction solution was stopped. The reaction solution was poured into 100mL of water, and filtered. The filter cake was dried to obtain the titlecompound7-(2-fluoro-6-(trifluoromethyl)benzyl)-5-(6-methoxypyridazin-3-yl)-3-methyl-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione 12c (465 mg,yellow solid), yield: 81.4%.

MS m/z (ESI): 570.0 [M−1]

Step 43-(bromomethyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-5-(6-methoxypyridazin-3-yl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

7-(2-fluoro-6-(trifluoromethyl)benzyl)-5-(6-methoxypyridazin-3-yl)-3-methyl-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione 12c (465 mg, 0.81mmol), azobisisobutyronitrile (13 mg, 0.081 mmol), N-bromosuccinimide(174 mg, 0.97 mmol), and 20 mL of chlorobenzene were added to a 100 mLreaction flask. The mixture was reacted for 17 hours at 80° C. underargon atmosphere, then the reaction was stopped. The reaction solutionwas concentrated under reduced pressure to obtain the crude titlecompound3-(bromomethyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-5-(6-methoxypyridazin-3-yl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione12d (600 mg, yellow oil), which was used directly in the next step.

MS m/z (ESI): 650.1 [M+1]

Step 53-((dimethylamino)methyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-5-(6-methoxypyridazin-3-yl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

The crude3-(bromomethyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-5-(6-methoxypyridazin-3-yl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione12d (15 mg, 0.033 mmol) was dissolved in 20 mL of THF, and added with a2M solution of dimethylamine in THF (1 mL, 2.03 mmol). The mixture wasreacted for 2 hours at room temperature, and then the reaction wasstopped. The reaction solution was concentrated under reduced pressure.The residues were purified by thin layer chromatography with elutionsystem A to obtain the title compound3-((dimethylamino)methyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-5-(6-methoxypyridazin-3-yl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione12e (85 mg, yellow solid), yield: 17.0%.

MS m/z (ESI): 615.2 [M+1]

Step 62-(4-aminophenyl)-3-((dimethylamino)methyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-5-(6-methoxypyridazin-3-yl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

3-((dimethylamino)methyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-5-(6-methoxypyridazin-3-yl)-2-(4-nitrophenyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione12e (100 mg, 0.14 mmol) was dissolved in 5 mL of formic acid, and addedwith 10% palladium on carbon (48 mg). The mixture was purged withhydrogen under normal pressure three times, and then reacted for 3 hoursat room temperature under hydrogen atmosphere. The reaction was stoppedand filtered. The filtrate was poured into 10 mL of water, and thenadded dropwise with saturated sodium bicarbonate solution to adjust thepH>7. The solution was extracted with dichloromethane (25 mL×3). Theorganic phases were combined, dried over anhydrous sodium sulfate, andfiltered. The filtrate was concentrated under reduced pressure and theresidue was purified by thin layer chromatography with a elution systemA to obtain the title compound2-(4-aminophenyl)-3-((dimethylamino)methyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-5-(6-methoxypyridazin-3-yl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione12f (50 mg, white solid), yield: 53.0%.

MS m/z (ESI): 585.2 [M+1]

Step 71-(4-(3-((dimethylamino)methyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-5-(6-methoxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methylurea

Acetic acid (51 mg, 085 mmol) and N,N-diisopropylethylamine (0.22 mL,1.28 mmol) were dissolved in 2 mL of toluene, and added withdiphenylphosphoryl azide (236 mg, 0.86 mmol). After reacting for 1.5hours at 75° C., the reaction solution was added with a 2 mL solution of2-(4-aminophenyl)-3-((dimethylamino)methyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-5-(6-methoxypyridazin-3-yl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione12f (15 mg, 0.033 mmol) in THF. The resulting solution was reacted for12 hours at 50° C., and then the reaction was stopped. The reactionsolution was added with 10 mL of saturated sodium bicarbonate solution,and then extracted with dichloromethane (20 mL×1). The organic phase wascollected and washed with water (10 mL×1) and saturated sodium chloride(10 mL×1), successively. The organic phase was concentrated underreduced pressure. The residue was purified by thin layer chromatographywith elution system A to obtain the title compound1-(4-(3-((dimethylamino)methyl)-7-(2-fluoro-6-(trifluoromethyl)benzyl)-5-(6-methoxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methylurea12 (20 mg, white solid), yield: 36.3%.

MS m/z (ESI): 642.3 [M+1]

¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 7.56 (d, 1H), 7.43-7.46 (m, 3H),7.34-7.39 (m, 3H), 7.27 (d, 1H), 7.16 (t, 1H), 6.05 (s, 1H), 5.48 (s,2H), 4.22 (s, 3H), 3.48 (s, 2H), 2.93 (d, 3H), 2.21 (s, 6H).

Example 131-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-ethylurea

Step 1 (E)-4-(dimethylamino)-3-(4-nitrophenyl)but-3-en-2-one

1-(4-nitrophenyl)propan-2-one 13a (3.8 g, 21.21 mmol) and N,N-dimethylformamide dimethyl acetal were added to a 50 mL flask. After reactingfor 30 minutes at 100° C., the reaction was stopped and added with 150mL of dichloromethane under stirring. The resulting product was purifiedby silica gel column chromatography with eluent system A to obtain thetitle compound (E)-4-(dimethylamino)-3-(4-nitrophenyl)but-3-en-2-one 13b(3 g, yellow solid), yield: 60.5%.

MS m/z (ESI): 235.0 [M+1]

Step 2 Ethyl 3-methyl-4-(4-nitrophenyl)-1H-pyrrole-2-carboxylate

(E)-4-(dimethylamino)-3-(4-nitrophenyl) but-3-en-2-one 13b (1.50 g, 6.48mmol) and 2-aminoacetylacetatehydrochloride (1.40 g, 7.78 mmol) weredissolved in 40 mL of acetic acid. The mixture was reacted at roomtemperature for 16 hours, and at 100° C. for 5 hours, then the reactionwas stopped. The reaction solution was concentrated under reducedpressure. The residue was added with 120 mL of dichloromethane, andwashed with saturated sodium bicarbonate solution (50 mL×1). The organicphase was dried over anhydrous sodium sulfate, and filtered. Thefiltrate was concentrated under reduced pressure, and the residue wasslurried with 15 mL of ethylacetate, and then filtered. The filter cakewas dried to obtain the title compound ethyl3-methyl-4-(4-nitrophenyl)-1H-pyrrole-2-carboxylate 13c (412 mg, yellowsolid), yield: 24.5%.

MS m/z (ESI): 273.1 [M−1]

Step 3 Ethyl 1-amino-3-methyl-4-(4-nitrophenyl)-1H-pyrrole-2-carboxylate

Ethyl 3-methyl-4-(4-nitrophenyl)-1H-pyrrole-2-carboxylate 13c (412 mg,1.50 mmol) was dissolved in 6 mL of N,N-dimethyl formamide, and thenadded with sodium hydride (72 mg, 1.80 mmol) in an ice bath. The mixturewas reacted for 30 minutes at room temperature, and then added with 20mL of chloramines solution. The mixture was further reacted for 2 hoursat room temperature and then the reaction was stopped. The reactionsolution was added with 50 mL of saturated sodium thiosulfate solution,stirred for 10 minutes, and left to separate into layers. The aqueousphase was extracted with ether (30 mL×3). The organic phase was washedwith saturated sodium chloride solution (100 mL×2), dried over anhydroussodium sulfate, and filtered. The filtrate was concentrated underreduced pressure to obtain the title compound ethyl1-amino-3-methyl-4-(4-nitrophenyl)-1H-pyrrole-2-carboxylate 13d (381 mg,yellow solid), yield 87.7%.

MS m/z (ESI): 290.2 [M+1]

Step 4 Ethyl1-(3-(2-fluoro-3-methoxyphenyl)ureido)-3-methyl-4-(4-nitrophenyl)-1H-pyrrole-2-carboxylate

Ethyl 1-amino-3-methyl-4-(4-nitrophenyl)-1H-pyrrole-2-carboxylate 13d(381 mg, 1.30 mmol) was added to 30 mL of dichloromethane and stirreduntil dissolution, then added with triethylamine (328 mg, 3.25 mmol) andtriphosgene (157 mg, 0.53 mmol). After reacting for 30 minutes at roomtemperature, the reaction solution was added with2-fluoro-3-methoxyaniline (202 mg, 1.43 mmol). The mixture was furtherreacted at room temperature for 2 hours. The reaction was stopped. Thereaction solution was filtered. The filter cake was washed with water(10 mL×2) and ethylacetate (2 mL×3), successively, and then dried toobtain the title compound ethyl1-(3-(2-fluoro-3-methoxyphenyl)ureido)-3-methyl-4-(4-nitrophenyl)-1H-pyrrole-2-carboxylate13e (296 mg, yellow solid), yield 49.9%.

MS m/z (ESI): 457.3 [M+1]

Step 53-(2-fluoro-3-methoxyphenyl)-5-methyl-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione

Ethyl1-(3-(2-fluoro-3-methoxyphenyl)ureido)-3-methyl-4-(4-nitrophenyl)-1H-pyrrole-2-carboxylate13e (290 mg, 0.64 mmol) and sodium hydroxide (128 mg, 3.20 mmol) weredissolved in 10 mL of dichloromethane. After reacting for 2 hours at 80°C., the reaction was stopped. The reaction solution was cooled to roomtemperature, and then added dropwise with 1 M hydrochloric acid solutionto adjust the pH to 3 to 4. A solid was separated and filtered. Thefilter cake was washed with water (20 mL×3) and ethanol (10 mL×1),successively to obtain the title compound3-(2-fluoro-3-methoxyphenyl)-5-methyl-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione13f (192 mg, gray solid), yield 73.3%.

MS m/z (ESI): 411.0 [M+1]

Step 61-(2,6-difluorobenzyl)-3-(2-fluoro-3-methoxyphenyl)-5-methyl-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione

3-(2-fluoro-3-methoxyphenyl)-5-methyl-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione13f (192 mg, 0.47 mmol), 2-(chloromethyl)-1,3-difluorobenzene (84 mg,0.52 mmol) and potassium carbonate (162 mg, 1.75 mmol) were added to 10mL of N,N-dimethyl formamide. After reacting for 16 hours at 50° C., thereaction was stopped. The reaction solution was concentrated underreduced pressure. The residue was added with 20 mL of water, (20 mL×1)and extracted with dichloromethane (20 mL×1). The aqueous phase wasextracted with dichloromethane (10 mL×3). The organic phases werecollected, dried over anhydrous sodium sulfate, and filtered. Thefiltrate was concentrated under reduced pressure, and the residue waspurified by thin layer chromatography with elution system A to obtainthe title compound1-(2,6-difluorobenzyl)-3-(2-fluoro-3-methoxyphenyl)-5-methyl-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione13g (160 mg, yellow solid), yield 63.7%.

MS m/z (ESI): 537.0 [M+1]

Step 75-(bromomethyl)-1-(2,6-difluorobenzyl)-3-(2-fluoro-3-methoxyphenyl)-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione

1-(2,6-difluorobenzyl)-3-(2-fluoro-3-methoxyphenyl)-5-methyl-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione13g (120 mg, 0.22 mmol), azobisisobutyronitrile (8 mg, 0.044 mmol) andN-bromosuccinimide (47 mg, 0.26 mmol) were dissolved in 5 mL ofchlorobenzene. The mixture was reacted for 16 hours at 85° C. underargon atmosphere, and then the reaction was stopped. The reactionsolution was cooled to room temperature and concentrated under reducedpressure. The residue was added with 10 mL of water, and extracted withdichloromethane (10 mL×1). The aqueous phase was extracted withdichloromethane (10 mL×3). The organic phases were combined, washed withsaturated sodium chloride solution (20 mL×1), dried over anhydroussodium sulfate, and filtered. The filtrate was concentrated underreduced pressure to obtain the title compound5-(bromomethyl)-1-(2,6-difluorobenzyl)-3-(2-fluoro-3-methoxyphenyl)-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione13h (130 mg, yellow solid), yield: 96.2%.

MS m/z (ESI): 616.0 [M+1]

Step 81-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione

The crude5-(bromomethyl)-1-(2,6-difluorobenzyl)-3-(2-fluoro-3-methoxyphenyl)-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione13h (130 mg, 0.21 mmol) and N,N-diisopropylethylamine (81 mg, 0.63 mmol)were dissolved in 5 mL of THF, and added with 1.6 mL solution ofdimethylamine in tetrahydrofuran. The mixture was reacted for 1 hour at0 to 5° C., and then the reaction was stopped. The reaction solution wasconcentrated under reduced pressure, and the residue was purified bythin layer chromatography with elution system A to obtain the titlecompound1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione13i (40 mg, yellow solid), yield: 33.1%.

MS m/z (ESI): 580.2 [M+1]

Step 96-(4-aminophenyl)-1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione

1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione131 (40 mg, 0.069 mmol), ammonium chloride (30 mg, 0.55 mmol), and iron(16 mg, 0.28 mmol) were dissolved in 5 mL mixture of ethanol and water(V/V=4:1). The mixture was reacted for 1 hour at 80° C. and then thereaction was stopped. The reaction solution was added with 20 mL ofwater, and then extracted with dichloromethane (10 mL×3). The organicphases were combined, washed with saturated sodium chloride solution (20mL×1), dried over anhydrous sodium sulfate, and filtered. The filtratewas concentrated under reduced pressure and the residue was purified bythin layer chromatography with elution system A to obtain the titlecompound6-(4-aminophenyl)-1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione13j (20 mg, yellow solid), yield: 52.6%.

MS m/z (ESI): 550.2 [M+1]

Step 101-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-ethylurea

6-(4-aminophenyl)-1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione13j (20 mg, 0.036 mmol) and ethyl isocyanate (13 mg, 0.18 mmol) weredissolved in 5 mL of tetrahydrofuran in a 30 mL sealed tube. Afterreacting for 16 hours at 50° C., the reaction was stopped. The reactionsolution was concentrated under reduced pressure, and the residue waspurified by thin layer chromatography with elution system A to obtainthe title compound1-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-ethylurea13 (15 mg, white solid), yield: 68.1%.

MS m/z (ESI): 621.3 [M+1]

¹H NMR (400 MHz, DMSO-d₆) δ 7.87-7.77 (m, 4H), 7.69 (s, 1H), 7.58-7325(m, 4H), 7.12-7.08 (m, 1H), 6.80-6.71 (m, 1H), 4.42 (s, 2H), 3.83 (s,3H), 3.45-3.50 (m, 2H), 3.20-3.31 (m, 2H), 2.16 (s, 6H), 1.12-1.03 (m,3H)

Example 141-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-methylurea

Step 11-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-methylurea

Acetic acid (70 mg, 1.12 mmol) and N,N-diisopropylethylamine (0.32 mL,1.80 mmol) were dissolved in 1 mL of toluene, and then added withdiphenylphosphoryl azide (0.25 mL, 1.20 mmol). After reacting for 1.5hours at 70° C., the reaction solution was added with a 1 mL solution of6-(4-aminophenyl)-1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione13j (30 mg, 0.054 mmol) in dichloromethane. The reaction solution wasfurther reacted for 12 hours at room temperature, and then added with 15mL of water, and extracted with dichloromethane (30 mL×1). The organicphase was washed with saturated sodium bicarbonate (10 mL×1), water (10mL×1), and saturated sodium chloride solution (10 mL×1), successively,dried over anhydrous sodium sulfate, and filtered. The filtrate wasconcentrated under reduced pressure and the residue was purified by thinlayer chromatography with elution system A to obtain the title compound1-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-methylurea14 (9 mg, white solid), yield: 27.3%.

MS m/z (ESI): 606.9 [M+1]

¹H NMR (400 MHz, CDCl₃) δ 8.86 (s, 1H), 7.55 (s, 2H), 7.24-7.35 (m, 3H),7.68-7.14 (m, 6H), 6.21 (s, 1H), 5.60-5.74 (m, 2H), 4.54-4.59 (m, 2H),3.97 (s, 3H), 2.77 (s, 3H), 2.55 (s, 6H).

Example 151-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-methoxyurea

Step 11-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-methoxyurea

6-(4-aminophenyl)-1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione13j (55 mg, 0.10 mmol) was dissolved in 4 mL of tetrahydrofuran, andthen added with 4-nitrophenyl chloroformate (30 mg, 0.15 mmol) andpyridine (32 mg, 0.40 mmol). After reacting for 3 hours at 30° C., thereaction solution was then added with methoxylamine hydrochloride (25mg, 0.30 mmol), and reacted for 8 hours under sealing. The reaction wasstopped, and the reaction solution was concentrated under reducedpressure. The residues were purified by thin layer chromatography withelution system A for three times to obtain the title compound1-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(2-fluoro-3-methoxyphenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-methoxyurea15 (5 mg, yellow solid), yield: 8.1%.

MS m/z (ESI): 623.4 [M+1]

¹H NMR (400 MHz, DMSO-d₆) δ 7.90-7.87 (m, 2H), 7.85-7.80 (m, 2H), 7.60(s, 1H), 7.55-7.25 (m, 4H), 7.09-7.02 (m, 1H), 6.71-6.65 (m, 1H), 4.50(s, 2H), 3.84 (s, 3H), 3.67 (s, 3H), 3.54 (s, 2H), 2.15 (s, 6H)

Example 161-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-methylurea

Step 1 Ethyl3-methyl-4-(4-nitrophenyl)-1H-pyrrole-1-tert-butylcarboxylate-2-carboxylate

Ethyl 3-methyl-4-(4-nitrophenyl)-1H-pyrrole-2-carboxylate 13c (4.30 g,15.70 mmol) was dissolved in 200 mL of dichloromethane, and added withtriethylamine (21.7 mL, 157 mmol), 4-dimethylaminopyridine (192 mg, 1.57mmol) and di-tert-butyl dicarbonyl (5.14 g, 23.50 mmol), successively.After reacting for 12 hours at room temperature, the reaction wasstopped. The reaction solution was concentrated under reduced pressure.The residue was added with 100 mL of water, and then extracted withdichloromethane (50 mL×4). The organic phases were combined, washed withsaturated sodium chloride solution (150 mL×1), dried over anhydroussodium sulfate, and filtered. The filtrate was concentrated underreduced pressure and the residue was purified by silica gel columnchromatography with eluent system B to obtain the title compound ethyl3-methyl-4-(4-nitrophenyl)-1H-pyrrole-1-tert-butylcarboxylate-2-carboxylate16b (4 g, yellow solid), yield: 68.1%.

MS m/z (ESI): 273.1 [M−100]

Step 2 Ethyl3-bromomethyl-4-(4-nitrophenyl)-1H-pyrrole-1-tert-butylcarboxylate-2-carboxylate

Ethyl3-methyl-4-(4-nitrophenyl)-1H-pyrrole-1-tert-butylcarboxylate-2-carboxylate16b (4 g, 10.70 mmol), azobisisobutyronitrile (351 mg, 2.14 mmol) andN-bromosuccinimide (2.28 g, 12.80 mmol) were dissolved in 40 mL ofchlorobenzene, successively. The reaction solution was reacted for 16hours at 80° C. under argon atmosphere. The reaction was stopped andcooled to room temperature. The reaction solution was concentrated underreduced pressure, and dried in vacuo to obtain the title compound ethyl3-bromomethyl-4-(4-nitrophenyl)-1H-pyrrole-1-tert-butylcarboxylate-2-carboxylate16e (5.2 g, yellow solid), which was used directly in the next step.

MS m/z (ESI): 453.0 [M−100]

Step 3 Ethyl3-(dimethylamino)methyl-4-(4-nitrophenyl)-1H-pyrrole-1-tert-butylcarboxylate-2-carboxylate

Ethyl3-bromomethyl-4-(4-nitrophenyl)-1H-pyrrole-1-tert-butylcarboxylate-2-carboxylate16c (4.80 g, 10.70 mmol) was dissolved in 50 mL of tetrahydrofuran, andadded with a solution of dimethylamine in tetrahydrofuran (26 mL, 53.50mmol). After reacting for 3 hours at room temperature, the reaction wasstopped. The reaction solution was concentrated under reduced pressure,and the residue was purified by silica gel column chromatography witheluant system A to obtain the title compound ethyl3-(dimethylamino)methyl-4-(4-nitrophenyl)-1H-pyrrole-1-tert-butylcarboxylate-2-carboxylate16d (3 g, yellow solid), yield: 67.4%.

MS m/z (ESI): 317.0 [M−100]

Step 4 Ethyl 3-((dimethylamino)methyl)-4-(4-nitrophenyl)-1H-pyrrole-2-carboxylate

Ethyl3-(dimethylamino)methyl-4-(4-nitrophenyl)-1H-pyrrole-1-tert-butylcarboxylate-2-carboxylate16d (3 g, 7.18 mmol) was dissolved in a 30 mL solution of 2 Mhydrochloric acid in methanol. After reacting for 3 hours at roomtemperature, the reaction was stopped, and the reaction solution wasconcentrated under reduced pressure. The residue was added with 30 mL ofsaturated sodium carbonate solution, and extracted with dichloromethane(30 mL×4). The organic phases were combined, washed with saturatedsodium chloride solution (60 mL×1), dried over anhydrous sodium sulfate,and filtered. The filtrate was concentrated under reduced pressure toobtain the title compound ethyl3-((dimethylamino)methyl)-4-(4-nitrophenyl)-1H-pyrrole-2-carboxylate 16e(2 g, yellow solid), yield: 90.9%.

MS m/z (ESI): 318.1 [M+1]

Step 5 Ethyl1-amino-3-((dimethylamino)methyl)-4-(4-nitrophenyl)-1H-pyrrole-2-carboxylate

Ethyl3-((dimethylamino)methyl)-4-(4-nitrophenyl)-1H-pyrrole-2-carboxylate 16e(1.8 g, 5.68 mmol) was dissolved in 20 mL of N,N-dimethylformamide, andadded with 60% sodium hydroxide (307 mg, 7.66 mmol). After reacting for30 minutes at room temperature, the reaction mixture was added with 50mL of chloramine, and further reacted for 2 hours at room temperature.The reaction was stopped and the reaction solution was added with 200 mLof water, and extracted with ether (50 mL×3). The organic phases werecombined, washed with saturated sodium chloride solution (100 mL×1),dried over anhydrous sodium sulfate, and filtered. The filtrate wasconcentrated under reduced pressure to obtain the title compound ethyl1-amino-3-((dimethylamino)methyl)-4-(4-nitrophenyl)-1H-pyrrole-2-carboxylate16f (620 mg, yellow solid), yield: 32.9%.

MS m/z (ESI): 333.2 [M+1]

Step 6 Ethyl3-((dimethylamino)methyl)-1-(3-(6-methoxypyridazin-3-yl)ureido)-4-(4-nitrophenyl)-1H-pyrrole-2-carboxylate

Ethyl1-amino-3-((dimethylamino)methyl)-4-(4-nitrophenyl)-1H-pyrrole-2-carboxylate16f (420 mg, 1.26 mmol) was dissolved in 15 mL of dichloromethane, andthen added with triethylamine (382 mg, 3.78 mmol), triphosgene (131 mg,0.44 mmol) and 3-amino-6-methoxypyridazine (236 mg, 1.89 mmol),successively. After reacting for 2 hours at room temperature, thereaction was stopped and the reaction solution was concentrated underreduced pressure. The residue was purified by thin layer chromatographywith elution system A to obtain the title compound ethyl3-((dimethylamino)methyl)-1-(3-(6-methoxypyridazin-3-yl)ureido)-4-(4-nitrophenyl)-H-pyrrole-2-carboxylate16g (298 mg, yellow solid), yield: 30.2%.

Step 75-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione

Ethyl3-((dimethylamino)methyl)-1-(3-(6-methoxypyridazin-3-yl)ureido)-4-(4-nitrophenyl)-H-pyrrole-2-carboxylate16g (260 mg, 0.54 mmol) was dissolved in 10 mL of methanol, and thenadded with sodium methoxide (151 mg, 2.69 mmol). After reacting for 3hours at 50° C., the reaction was stopped and the reaction solution wasconcentrated under reduced pressure. The residue was purified by thinlayer chromatography with elution system A to obtain the title compound5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione16h (76 mg, yellow solid) yield: 32.3%.

MS m/z (ESI): 438.3 [M+1]

Step 81-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione

5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione16h (78 mg, 0.18 mmol), 2-chloromethyl-1,3-difluorophenyl (35 mg, 0.21mmol) and potassium carbonate (37 mg, 0.26 mmol) were dissolved in 5 mLof N,N-dimethylformamide. After reacting for 16 hours at 50° C., thereaction was stopped and the reaction solution was concentrated underreduced pressure. The residue was purified by thin layer chromatographywith elution systems A to obtain the title compound1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione16i (72 mg, yellow solid), yield: 72.0%.

MS m/z (ESI): 564.1 [M+1]

Step 96-(4-aminophenyl)-1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione

1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)-6-(4-nitrophenyl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione16i (72 mg, 0.13 mmol), iron (29 mg, 0.51 mmol), and ammonium chloride(55 mg, 1.02 mmol) were dissolved in a 20 mL mixture of ethanol andwater (V/V=4:1). After reacting for 1 hour at 80° C., the reaction wasstopped. The reaction solution was cooled to room temperature, and thenadded with 20 mL of water, and extracted with dichloromethane (10 mL×4).The organic phases were combined, washed with saturated sodium chloride(10 mL×1), dried over anhydrous sodium sulfate, and filtered. Thefiltrate was concentrated under reduced pressure. The residue waspurified by thin layer chromatography with elution system A to obtainthe title compound6-(4-aminophenyl)-1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione16j (25 mg, khaki solid), yield: 42.8%.

MS m/z (ESI): 534.3[M+1]

Step 101-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-methylurea

Acetic acid (51 kg, 0.84 mmol) was added to a 25 mL sealed tube, andadded with 2 mL of toluene, N,N-diisopropylethylamine (163 mg, 1.26mmol), and diphenylphosphoryl azide (231 mg, 0.84 mmol), successively.The mixture was reacted for 1 hour in the sealed tube. After coolingdown to 0° C., the reaction solution was added with 2 mL solution of6-(4-aminophenyl)-1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione16j (45 mg, 0.084 mmol) in dichloromethane, and further reacted for 16hours at 40° C. in the sealed tube. The reaction was stopped, and thereaction solution was concentrated under reduced pressure. The residuewas purified by thin layer chromatography with elution system A toobtain the title compound1-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-methylurea 16 (18 mg, white solid), yield: 36.0%.

MS m/z (ESI): 591.3 [M+1]

¹H NMR (400 MHz, DMSO-d₆) δ 7.92-7.87 (m, 2H), 7.77-7.70 (m, 2H), 7.69(s, 1H), 7.60-7.55 (m, 1H), 7.25-7.13 (m, 2H), 7.05-6.99 (m, 1H),6.79-6.71 (m, 1H), 4.48 (s, 2H), 4.06 (s, 3H), 3.54 (s, 2H), 2.75 (s,3H), 2.16 (s, 6H)

Example 171-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-hydroxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxyurea

Step 11-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-hydroxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxyurea

1-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-methoxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxyurea11 (122 mg, 0.20 mmol) was dissolved in 6 mL of tetrahydrofuran andstirred until it was dissolved completely. The solution was added with40% hydrobromic acid (103 mg, 0.51 mmol), and stirred for 12 hours atroom temperature. The reaction was stopped, and the reaction solutionwas added with 5 mL of saturated sodium bicarbonate solution and furtherstirred for 5 minutes. The resulting mixture was extracted. The organicphases were combined and purified by silica gel column chromatographywith elution systems A to obtain the title compound1-(4-(7-(2,6-difluorobenzyl)-3-((dimethylamino)methyl)-5-(6-hydroxypyridazin-3-yl)-4,6-dioxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxyurea17 (16 mg, white solid), yield: 13.5%.

MS m/z (ESI): 594.3 [M+1]

¹H NMR (400 MHz, DMSO-d₆) δ 13.36-13.18 (m, 1H), 9.69 (s, 1H), 9.21-9.14(m, 1H), 7.83-7.76 (m, 2H), 7.71-7.64 (m, 2H), 7.52-7.46 (m, 11H),7.46-7.36 (m, 1H), 7.09 (s, 3H), 5.32-5.21 (m, 2H), 3.71-3.62 (m, 5H),2.17 (s, 6H)

Example 181-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-methoxyurea

Step 11-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-methoxyurea

6-(4-aminophenyl)-1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione16j (170 mg, 0.319 mmol), methyl 4-nitrophenylmethoxyamino formate (81mg, 0.382 mmol, prepared by a known method disclosed in “WO 2011090935”)and N,N-diisopropylethylamine (109 μL, 0.638 mmol) were dissolved in 10mL of tetrahydrofuran. After reacting for 4 hours at room temperature,the reaction was stopped and the reaction solution was concentratedunder reduced pressure. The residue was purified by thin layerchromatography with elution system A to obtain the title compound1-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrrolo[2,1-f][1,2,4]triazin-6-yl)phenyl)-3-methoxyurea18 (15 mg, yellow solid), yield: 7.8%.

MS m/z (ESI): 607.2 [M+1]

Test Examples Biological Evaluation Test Example 1. Human GnRHr (GnRHReceptor) Activity Assay of the Present Compounds

In vitro GnRHr protein activity was tested by the following methods.

This assay was used to determine the inhibition effect of the presentcompound on the activity of human GnRHr protein expressed by HumanGnRHr/CHO stably transfected cell lines.

1. Experimental Materials and Equipments

1) Fluo-4 NW Calcium Assay Kits (F36206, Invitrogen)

2) DMEM/F12 (SH30023.01B, Thermo)

3) G418 (11811-031, Invitrogen)

4) FlexStation3 Microplate Reader

2. Experimental Protocol

A mammalian expression vector containing the human GnRHr gene wastransferred into CHO cells by adding Lipofectamine®LTX reagentcontaining Plus™. Antibiotics were added the next day for screening topick out the monoclonal cell lines.

The Human GnRHr/CHO stably transfected cell lines were inoculated in96-well plates with an inoculation density of 25,000 cells/well. Theculture medium was removed the next day, and loading buffer containingFluo-4 dye was added to the plate (100 μL/well) and incubated for 30minutes at 37° C. The plate was moved to room temperature andequilibrated for 10 minutes. Each compound was diluted with DMSO toseven concentration gradients of 100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM,0.001 μM, 0.0001 μM. Then, 1 μl of each gradient was added to each welland incubated for 10 minutes at room temperature. After automatedaddition of 50 μL of GnRH polypeptide stimulant solution, the value wasimmediately detected at 494/516 nM by a microplate reader (flexstation3). IC₅₀ values of the compounds were calculated by software fromdifferent fluorescence signals at various corresponding concentrations.

The inhibitory activity of the present compounds on human GnRHr wasdetermined by the above assay, and IC₅₀ values are shown in Table 1.

TABLE 1 Inhibition effect (IC₅₀) of the present compounds on theactivity of Human GnRHr Example No. IC₅₀ (nM) 2 1.46 3 0.90 5 0.66 60.96 7 0.49 8 0.60 9 0.42 10 0.66 11 4.95 12 2.24 13 0.92 14 0.11 150.43 16 2.32 Conclusion: The present compounds have significant humanGnRHr inhibitory activity.

Test Example 2. Monkey GnRHr Inhibitory Activity Assay of the PresentCompounds

This assay was used to determine the inhibition effect of the presentcompounds on the activity of monkey GnRHr protein expressed by monkeyGnRHr/CHO stably transfected cell lines.

1. Experimental Materials and Equipments

1) Fluo-4 NW Calcium Assay Kits (F36206, Invitrogen)

2) DMEM/F12 (SH30023.01B, Thermo)

3) G418 (11811-031, Invitrogen)

4) FlexStation3 Microplate Reader

2. Experimental Protocol

A mammalian expression vector containing the monkey GnRHr gene wastransferred into CHO cells by adding Lipofectamine®LTX reagentcontaining Plus™. Antibiotics were added the next day for screening topick out the monoclonal cell lines.

The monkey GnRHr/CHO stably transfected cell lines were inoculated in96-well plates with an inoculation density of 25,000 cells/well. Theculture medium was removed the next day, and loading buffer containingFluo-4 dye was added to the plate (100 L/well) and incubated for 30minutes at 37° C. The plate was moved to room temperature andequilibrated for 10 minutes. Each compound was diluted with DMSO toseven concentration gradients of 100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM,0.001 μM, 0.0001 μM. Then, 1 μl of each gradient was added to each welland incubated for 10 minutes at room temperature. After automatedaddition of 50 μL of GnRH polypeptide stimulant solution, the value wasimmediately detected at 494/516 nM by a microplate reader (flexstation3). IC₅₀ values of the compounds were calculated by software fromdifferent fluorescence signals at various corresponding concentrations.

The inhibitory activity of the present compounds on monkey GnRHr wasdetermined by the above assay, and IC₅₀ values are shown in Table 1.

TABLE 2 Inhibition effect (IC₅₀) of the present compounds on theactivity of Monkey GnRHr Example No. IC₅₀ (nM) 9 23.22 11 78.57Conclusion: The present compounds have significant monkey GnRHrinhibitory activity.

Test Example 3. Rabbit GnRHr Inhibitory Activity Assay of the PresentCompounds

This assay was used to determine the inhibition effect of the presentcompounds on the activity of rabbit GnRHr protein expressed by rabbitGnRHr/CHO stably transfected cell lines.

1. Experimental Materials and Equipments

1) Fluo-4 NW Calcium Assay Kits (F36206, Invitrogen)

2) DMEM/F12 (SH30023.01B, Thermo)

3) G418 (11811-031, Invitrogen)

4) FlexStation3Microplate Reader

2. Experimental Protocol

A mammalian expression vector containing the rabbit GnRHr gene wastransferred into CHO cells by adding Lipofectamine®LTX reagentcontaining Plus™. Antibiotics were added the next day for screening topick out the monoclonal cell lines.

The rabbit GnRHr/CHO stably transfected cell lines were inoculated in96-well plates with an inoculation density of 25,000 cells/well. Theculture medium was removed the next day, and loading buffer containingFluo-4 dye was added to the plate (100 μL/well) and incubated for 30minutes at 37° C. The plate was moved to room temperature andequilibrated for 10 minutes. Each compound was diluted with DMSO toseven concentration gradients of 100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM,0.001 μM, 0.0001 μM. Then, 1 μl of each gradient was added to each welland incubated for 10 minutes at room temperature. After automatedaddition of 50 μL of GnRH polypeptide stimulant solution, the value wasimmediately detected at 494/516n M by a microplate reader (flexstation3). IC₅₀ values of the compounds were calculated by software fromdifferent fluorescence signals at various corresponding concentrations.

The inhibitory activity of the present compounds on rabbit GnRHr wasdetermined by the above assay, and IC₅₀ values are shown in Table 1.

TABLE 3 IC₅₀ of inhibition effect of the present compound on rabbitGnRHr activity Example No. IC₅₀ (nM) 8 0.27 9 0.24 11 0.57 Conclusion:The present compounds have significant rabbit GnRHr inhibitory activity.

Pharmacokinetics Evaluation Test Example 4. Rat Pharmacokinetics Assayof the Compounds of Example 2, Example 3, Example 5, Example 9, Example10, Example 11, and Example 13 of the Present Invention

1. Summary

Sprague-Dawley (SD) rats were used as test animals. The plasmaconcentrations at various times were determined by LC/MS/MS afterintragastric administration of compounds of Example 2, Example 3,Example 5, Example 9, Example 10, Example 11, and Example 13, andintravenous administration of the compound of Example 11 to the rats.The pharmacokinetic behavior of the present compounds in rats wasstudied, and the pharmacokinetic features were evaluated.

2. Test Protocol

2.1 Samples

Compounds of Example 2, Example 3, Example 5, Example 9, Example 10,Example 11, and Example 13

2.2 Test Animals

Thirty-two (32) healthy adult SD rats (half female and half male) wereevenly designated into 8 groups (4 for each group). The animals werepurchased from Shanghai Super B&K Laboratory Animal Corp. Ltd(laboratory animal production Certificate No. SCXK (HU)2008-0016).

2.3 Drug Formulation

An appropriate amount of sample was weighed, and added with 0.5% CMC-Nato form a 1 mg/ml suspension upon ultrasonic treatment.

Appropriate amounts of drugs were weighed, and dissolved with 0.5 mlDMSO and 0.5 ml of Tween 80. Saline solution was added to the finalvolume.

2.4 Administration

Thirty-two (32) SD rats (half female and half male) were evenlydesignated into 8 groups (4 for each group), and administered by oralgavage or intravenous injection (i.v.) (oral gavage at a dosage of 10mg/kg or 30 mg/kg, and i.v. at a dosage of 5.0 mg/kg), respectively, ata volume of 10 ml/kg after fasting overnight.

3. Operation

0.1 ml of blood was sampled before, and 0.5, 1, 2, 4, 6, 8, 11, and 24hours after administration, collected in a heparinized test tube, andcentrifuged at 3500 rpm for 10 minutes to separate the plasma, which wasstored at −20° C. The animals were allowed access to feed 2 hours afteradministration.

The content of the test compound in the plasma of rats afteradministration of the compound by oral gavage was determined byLC/MS/MS.

4. Pharmacokinetic Parameters Results

Pharmacokinetic parameters of the present compounds are shown in thefollowing Table 4:

Rat Pharmacokinetic test (10 mg/kg) Area Apparent Oral Plasma underResidence Clearance volume of Bioavailability concentration curveHalf-life time rate distribution Example F C_(max) AUC T_(1/2) MRT CL/FVz/F No. (%) (ng/mL) (ng/mL*h) (h) (h) (ml/min/kg) (ml/kg) 2 — 746 ± 4931546 ± 650  1.24 ± 0.54 2.02 ± 0.67 121 ± 44  14406 ± 10470 3 — 237 ±158 1072 ± 472  6.17 ± 0.79 6.42 ± 1.39 181 ± 79  96163 ± 42245 5 — 690± 660 1290 ± 1028 2.32 ± 0.65 2.47 ± 0.75 247 ± 198 46774 ± 39154 9 —146 ± 59  628 ± 369 2.91 ± 1.64 4.67 ± 2.22 332 ± 159 72021 ± 24995 10 —598 ± 431 1237 ± 593  2.29 ± 0.84 2.96 ± 0.17 168 ± 95  30330 ± 14422 1176.6 2438 ± 873  4092 ± 1439 0.84 ± 0.18 1.56 ± 0.34 44.2 ± 13.4 3287 ±1512 (oral10 mg/kg) i.v. 2673 ± 1243 1.59 ± 0.50 2.25 ± 0.06 35.4 ± 12.54929 ± 2390 (5 mg/kg) 13 — 103 ± 64  389 ± 298 3.31 ± 0.57 4.48 ± 0.70 915 ± 1046 231402 ± 233610 Conclusion: The present compounds are wellabsorbed orally in rats, and have obvious pharmacokinetic absorption.Example 11 has particularly preferable oral bioavailability.

Test Example 5. Rabbit Pharmacokinetics Assay of Example 11 of thePresent Invention

1. Summary

Rabbits were used as test animals. The plasma concentrations at varioustimes were determined by LC/MS/MS after intragastric administration ofthe compound of Example 11, and intravenous administration of thecompound of Example 11 to rabbits. The pharmacokinetic behavior of thepresent compound in rabbits was studied, and the pharmacokineticfeatures were evaluated.

2. Test Protocol

2.1 Sample

Compound of Example 11

2.2 Test Animals

Six (6) healthy New Zealand Rabbits (female) were evenly designated into2 groups. The animals were purchased from Jie Sijie Corp. Ltd.

2.3 Drug Formulation

An appropriate amount of sample was weighed, and added with 0.5% CMC-Nato form 3 mg/ml suspension upon ultrasonic treatment.

Appropriate amounts of drugs were weighed, and dissolved with 1 ml DMSOand 1 ml of Tween 80. Saline solution was added to the final volume.

2.4 Administration

Six (6) healthy New Zealand rabbits (female) were evenly designated into2 groups, and administered by oral gavage or intravenous injection(i.v.) (oral gavage at a dosage of 30 mg/kg with an administrationvolume of 5 ml/kg, and i.v. at a dosage of 5 mg/kg with anadministration volume of 2 ml/kg) after fasting overnight.

3. Operation

0.2 ml of blood was sampled before and 5 minutes, 0.25, 0.5, 1, 2, 4, 8,11, 24 hours after intravenous administration, collected in aheparinized test tube, and centrifuged at 3500 rpm for 10 minutes toseparate the plasma, which was stored at −20° C. Blood was sampledbefore, and 0.5, 1, 2, 4, 6, 8, 11, and 24 hours after oraladministration, and treated the same as that of the intravenousadministration group. The content of the test compound in the plasma ofrabbits after administration of the compound by oral gavage andintravenous injection was determined by LC/MS/MS.

4. Pharmacokinetic Parameters Results

Pharmacokinetic parameters of the present compounds are shown in thefollowing Table 5:

Rabbit Pharmacokinetic test Apparent Oral Plasma Area under ResidenceClearance volume of Bioavailability concentration curve Half-life timerate distribution F C_(max) AUC T_(1/2) MRT CL/F Vz/F No (%) (ng/mL)(ng/mL*h) (h) (h) (ml/min/kg) (ml/kg) 11 92.9 5043 ± 776 19008 ± 23124.57 ± 1.06 3.94 ± 0.55 26.6 ± 3.4 10719 ± 3862 (oral 30 mg/kg)intravenous injection 3411 ± 832 4.60 ± 2.70 2.39 ± 0.30 25.3 ± 5.510948 ± 7749 (5 mg/kg) Conclusion: The compound of Example 11 of thepresent invention is well absorbed in rabbits, and has preferable oralbioavailability.

Test Example 6. Beagle Pharmacokinetics Assay of Example 11 of thePresent Invention

1. Summary

Beagles were used as test animals. The plasma concentrations at varioustimes were determined by LC/MS/MS after intragastric administration ofthe compound of Example 11, and intravenous administration of thecompound of Example 11 to beagles. The pharmacokinetic behavior of thepresent compound in beagles was studied, and the pharmacokineticfeatures were evaluated.

2. Test Protocol

2.1 Sample

Compound of Example 11

2.2 Test Animals

Eight (8) healthy beagles (half female and half male) were evenlydesignated into 2 groups. The animals were purchased from SuzhouXishanZhongke Laboratory Animal Corp. Ltd.

2.3 Drug Formulation

An appropriate amount of sample was weighed, and added with 0.5% CMC-Nato form a 1.0 mg/ml suspension upon ultrasonic treatment;

Appropriate amounts of drugs were weighed, and dissolved with 5 ml DMSOand 5 ml of Tween 80. Saline solution was added to the final volume.

2.4 Administration

Eight (8) healthy beagles (half female and half male) were evenlydesignated into 2 groups, and administered by oral gavage or intravenousinjection (i.v.) (oral gavage at a dosage of 5 mg/kg with anadministration volume of 5 ml/kg, and i.v. at a dosage of 2 mg/kg withan administration volume of 2 ml/kg) after fasting overnight.

3. Operation

1.0 ml of blood was sampled from the foreleg vein before, and 0.25, 0.5,1, 2, 4, 6, 8, 12, and 24 hours after oral administration, collected ina heparinized test tube, and centrifuged at 3500 rpm for 10 minutes toseparate the plasma, which was stored at −20° C. The animals wereallowed access to feed 2 hours after administration. Blood was sampledbefore, and 5 minutes, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours afterintravenous administration, and treated the same as above. The contentof the test compound in the plasma of beagles after administration ofthe compound by oral gavage and intravenous injection was determined byLC/MS/MS.

4. Pharmacokinetic Parameters Results

Pharmacokinetic parameters of the present compounds are shown in thefollowing Table 6:

Beagle Pharmacokinetic test Apparent Oral Plasma Area under ResidenceClearance volume of Bioavailability concentration curve Half-life timerate distribution F C_(max) AUC T_(1/2) MRT CL/F Vz/F No. (%) (ng/mL)(ng/mL*h) (h) (h) (ml/min/kg) (ml/kg) 11 103.5 1087 ± 350 5942 ± 13455.37 ± 1.52 6.40 ± 2.82 14.7 ± 4.2 7246 ± 4373 (oral 5 mg/kg) i.v. 2296± 213  3.34 ± 0.40 4.02 ± 0.50 14.6 ± 1.3 4235 ± 706  (2 mg/kg)Conclusion: The compound of Example 11 of the present invention is wellabsorbed in beagles, and has preferable oral bioavailability.

Test Example 7. In Vivo Pharmacodynamics Test of Example 11 of thePresent Invention in Rabbit

1. Summary

An endometriosis model was established in rabbits, and a pharmacodynamicscreening test for rabbit endometriosis treated by the compound ofExample 11 was implemented.

2. Test Protocol

2.1 Sample

Compound of Example 11

2.2 Test Animals

Fifteen (15) healthy New Zealand rabbits (female) were evenly designatedinto 3 groups. The animals were purchased from Shanghai SongjiangDistrict Songlian Laboratory Animal Farm (laboratory animal productionCertificate No. SCXK (HU) 2008-0016).

2.3 Operation

Rabbits were adaptive fed and quarantined for seven days, andsubcutaneously injected with 30 g/kg of estradiol benzoate once dailyfor two days before surgery. When the model was established, rabbitswere anesthetized with 3% sodium pentobarbital, and laparotomizedsterilely. Then, one side of the uterus was separated and ligated withthe uterine mesorectum vessel. Then, a section of the uterus was cut offand put into a warm saline dish, and then dissected longitudinally andthe myometrium and endometrium were separated. A section of endometrium(about 0.5 cm by 0.5 cm) was seamed to the abdominal wall rich in bloodvessels with the intimal surface facing toward the abdominal cavity.Three weeks after endometrium planting, the rabbits were laparotomizedto check graft intimal size. 15 animals of successful modeling wererandomly divided into 3 groups (5 for each group), which were vehiclecontrol group, low-dose group (10 mg/kg of the compound of Example 11),and high-dose group (30 mg/kg of the compound of Example 11),respectively. Each group was administered by oral gavage once a day in avolume of 5 ml/kg, continuously for 28 days after successful modeling.Ultimately, the rabbit was weighed and the volume of ectopic endometriumwas measured to evaluate the effect of the present compound on theendometrial activity.

2.4 Results:

After 30 mg/kg administration of the compound of Example 11 to therabbit endometriosis model for 4 weeks with oral administration of thesame amount of 0.5% CMC-Na as a control, the growth of ectopicendometrium was significantly inhibited, while the inhibition of thecompound of Example 11 for the ectopic endometrial volume at 10 mg/kgwas not obvious. Therefore, the toxicologic NOAEL dose (dose under whichno damage effect dose was observed) of the compound of Example 11 in therabbit endometriosis model is 30 mg/kg.

Test Example 8. Seven-Day Toxicokinetic Evaluation of the Compound ofExample 11 in Rats

1. Summary

SD rats were used as test animals. The plasma concentration at varioustimes was determined by LC/MS/MS after administration of the compound ofExample 11 (SHR147280) by oral gavage for 7 days. The compound toxicityin rodents was preliminarily assessed according to the potentialtoxicity reaction and severity level of the body.

2. Test Protocol

2.1 Sample

Compound of Example 11

2.2 Test Animals

Twenty-four (24) healthy adult SD rats (half female and half male) wereevenly designated into 4 groups (6 for each group). The animals werepurchased from Shanghai SLAC Laboratory Animal Corp. Ltd (laboratoryanimal production Certificate No. SCXK (HU) 2012-0002).

2.3 Drug Formulation

An appropriate amount of sample was accurately weighed, and added withsome 0.5% CMC-Na to form an uniform suspension upon ultrasonicatorgrinding treatment, and then diluted to the final volume. The testsample solutions on day 1 and day 7 were kept for the determination ofadministration concentration of toxicokinetics.

2.4 Administration

Repeat oral doses were 80, 240, and 720 mg/kg, and administrationconcentrations were 8, 24, and 72 mg/ml respectively. The dosing volumewas 10 ml/kg once daily for 7 days.

3. Operation

The blood was sampled on day 1 and day 7 after administration withsampling time before, and 1, 2, 4, 8 and 24 hours after oraladministration on day 1, as well as before, and 1, 2, 4, 8, 24 and 48hours after oral administration on day 7. The blood sample was collectedin a heparinized test tube, and centrifuged to separate the plasma,which was stored at −20° C. The content of the test compound in theplasma of rats after administration of the compound by oral gavage wasdetermined by LC/MS/MS.

4. Results

4.1. Impact on Clinical Signs

4.1.1 Death

Rat No. 1 in the 720 mg/kg toxicity group of the compound of Example 11died on day 7 before dosing. Rat No. 5 in the 720 mg/kg toxicokineticgroup of the compound of Example 11 died on day 7 before dosing, and No.6 died on day 9.

4.1.2 Clinical Observation

During the administration, compared with the vehicle control group, ratsin the 720 mg/kg toxicity group and toxicokinetic group all showedweight loss and poor mental state after administration of the compoundof Example 11 on day 6. Rats in the 720 mg/kg toxicity group andtoxicokinetic group after administration of the compound of Example 11on day 7 all showed food intake reduction, weight loss, lessenedactivity, poor mental state, dull back fur, hair stand, roachback,haemorrhage around the nose and eyes, diarrhea, and filthy crissum.

4.2. Impact on Body Weight

Compared with the vehicle control group, the weight change percentage ofthe high dose group exhibited a significant decrease (P<0.05) afteradministration of the compound of Example 11 on day 3 and day 7.

4.3 Impact on Food Intake

Compared with the vehicle control group, the food intake of the grouptreated with the compound SHR147280 at 720 mg/kg dose level wassignificantly reduced on day 3 and day 7.

4.4. Hematology Test Results

Compared with the vehicle control group, WBC (white blood cells), Neut(neutrophils relative value and absolute neutrophils count), Lymph(absolute lymphocyte count), EO (absolute eosinophils cell count), andRET (reticulocyte relative value and absolute reticulocyte count) ofrats in the 720 mg/kg dose group of the compound of Example 11 weresignificantly decreased (P<0.05).

4.5. Blood Biochemical Test Results

Compared with the vehicle control group, BUN (blood urea nitrogen), CHOL(total cholesterol), CREA (creatinine), TBIL (total bilirubin), TP(total protein), and AST (aspartate aminotransferase) of rats in the 720mg/kg dose group of the compound of Example 11 were significantlyincreased (P<0.05). Although there were no statistical differences inALT (alanine aminotransferase), the ALT of two animals was significantlyincreased. ALB (albumin) and ALP (alkaline phosphatase) weresignificantly decreased (P<0.05), whereas the ion detection result waslacking for the shortage of blood samples of the SHR 147280 720 mg/kgdose group. No other obvious toxicological abnormalities were detected.

4.6. Coagulation Parameters

Compared with the vehicle control group, rats in the 80 mg/kg dose groupand 240 mg/kg dose group of the compound of Example 11 did not show asignificant difference. Coagulation parameters result was lacking forthe shortage of blood samples of the SHR 147280 720 mg/kg dose group.

4.7. The Urine Test

Compared with the vehicle control group, the treatment groups showed noregularly abnormal changes in urine indexes.

4.8. General Pathology Results

Autopsy was generally visible. Compared with the vehicle control group,the 720 mg/kg dose group of the compound of Example 11 exhibited thymushemorrhage and atrophy; spleen atrophy and congestion; varying degreesof congestion in leaves of the liver with flaky grayed necrotic lesions;lung hyperemia and hemorrhage; adrenal gland hemorrhage and hypertrophy;ovary hemorrhage and congestion; gastrointestinal wall thinning; whitecontents in the stomach; and yellow contents in the small intestine,intestinal hemorrhage, colon rectal hemorrhage, and intestine mucoid. Inaddition, the dead animals showed cardiac necrotic lesions. The rest ofthe groups presented no visible abnormality.

4.9. Organ Coefficient Results

Compared with the vehicle control group, the absolute wet weight of thethymus, heart, lung and spleen in male and female rats of the 720 mg/kggroup of the compound of Example 11 was significantly decreased(p<0.05). In contrast, the absolute wet weight of the adrenal gland wassignificantly increased (p<0.05). The Relative organ coefficient of thethymus and spleen was significantly reduced (p<0.05), whereas therelative organ coefficient of the brain, liver, lung, kidney, andadrenal gland was significantly increased (p<0.05).

4.10. Toxicokinetic Parameters Results

Toxicokinetic parameters of female rats after intragastricadministration Group 80 mg/kg 240 mg/kg 720 mg/kg Parameter Day 1 Day 7Day 1 Day 7 Day 1 Day 7 t_(max)(h) 1.5 4.0 8.0 6.0 16.0 24.0C_(max)(ng/ml) 17800 19550 41500 51150 69300 138000 AUC_(0-t)(ng/ml*h)173185 282074 639265 790873 1546150 2838650 AUC_(0-∞)(ng/ml*h) 173968313468 690196 840450 — — t_(1/2)(h) 3.15 6.79 5.73 4.26 — —CLz/F(ml/min/kg) 7.71 — 5.82 — — — Vz/F(ml/kg) 2092 — 2871 — — —MRT_(0-∞)(h) 5.82 9.23 9.63 7.97 — — Note: “—” indicates that theparameter cannot be calculated for the lack of data.

Therefore, toxicology NOAEL dose of Example 11 is 240 mg/kg, and thelethal dose is 720 mg/kg. The potential toxic target organs were marrowhematopoietic system, thymus, heart, liver, lung, spleen, adrenal gland,ovary, and digestion system.

In conclusion: Comparing the pharmacokinetic test of Test Example 5,pharmacodynamic test of Test Example 7, and toxicologicalpharmacokinetic test of Test Example 8, the preliminary evaluation ofthe safety window of the compound in Example 11 was as follows:

NOAEL dose (mg/kg) (toxicological pharmacokinetic AUC (ng/ml * h) Testdose/ (in vivo exposure/ comparison pharmacodynamic dose)pharmacokinetic exposure) Example 11 240/30 = 8 840450/(19008 ± 2312) =39.4 or 50.3

We claim:
 1. A method for treating uterine leiomyoma, endometriosis, orprostate cancer, the method comprising administering to the subject apharmaceutical composition comprising an effective amount of a compoundof formula (II), or a tautomer, mesomer, racemate, enantiomer, ordiastereomer thereof, or mixture thereof, or a pharmaceuticallyacceptable salt thereof, and one or more pharmaceutically acceptablecarriers, diluents, or excipients:

wherein R¹ is selected from the group consisting of aryl and heteroaryl,wherein the aryl and heteroaryl are each independently and optionallyfurther substituted with one or more groups selected from the groupconsisting of halogen, cyano, nitro, alkyl, haloalkyl, hydroxyalkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl and —OR⁶; R² is alkyl,wherein the alkyl is further substituted with aryl, wherein the aryl isoptionally further substituted with one or more groups selected from thegroup consisting of halogen, alkyl, haloalkyl, and —OR⁶; R³ is aryl,wherein the aryl is optionally further substituted with one or moregroups selected from the group consisting of halogen, alkyl, haloalkyl,—OR⁶, —C(O)OR⁶, —OC(O)R⁶, —C(O)R⁶, —NHC(O)R⁶, —NHC(O)OR⁶, —NHC(O)NHR⁶,and —NHC(O)NHOR⁶; R⁴ is alkyl; and R⁵ is hydrogen; R⁶ is selected fromthe group consisting of hydrogen and alkyl; n is 1, 2, 3, or
 4. 2. Themethod according to claim 1, wherein the subject is in need of treatmentof prostate cancer.
 3. The method according to claim 1, wherein thesubject is in need of treatment of endometriosis.
 4. The methodaccording to claim 1, wherein the compound is selected from the groupconsisting of:


5. The method according to claim 1, wherein the subject is in need oftreatment of uterine leiomyoma.
 6. The method according to claim 2,wherein the compound is selected from the group consisting of:


7. The method according to claim 3, wherein the compound is selectedfrom the group consisting of:


8. The method according to claim 1, wherein the compound is


9. The method according to claim 5, wherein the compound is


10. The method according to claim 2, wherein the compound is


11. The method according to claim 3, wherein the compound is


12. A compound that is

or a tautomer, mesomer, racemate, enantiomer, or diastereomer thereof,or mixture thereof, or a pharmaceutically acceptable salt thereof. 13.The compound of claim 12, or a pharmaceutically acceptable salt thereof.14. The method according to claim 1, wherein R¹ is selected from phenylor pyridazinyl.
 15. The method according to claim 1, wherein R² isbenzyl, wherein the benzyl is optionally further substituted with one ormore groups selected from the group consisting of halogen, alkyl, —OR⁶and haloalkyl.
 16. The method according to claim 1, wherein R³ isphenyl, wherein the phenyl is optionally further substituted with one ormore groups selected from the group consisting of —NHC(O)R⁶, —NHC(O)OR⁶,—NHC(O)NHR⁶ and —NHC(O)NHOR⁶.
 17. The method according to claim 1,wherein R⁴ is methyl.
 18. The method according to claim 1, wherein n is1 or
 2. 19. The method according to claim 5, wherein the compound isselected from the group consisting of:


20. A pharmaceutical composition comprising the compound according toclaim 12, and one or more pharmaceutically acceptable carriers,diluents, or excipients.
 21. A pharmaceutical composition comprising thecompound according to claim 13, and one or more pharmaceuticallyacceptable carriers, diluents, or excipients.